Therapeutic TREM-1 peptides

ABSTRACT

A polypeptide comprising one or more sequences derived from CDR2 or CDR3 of a TREM-1 protein, characterized by the ability to treat, ameliorate, or lessen the symptoms of conditions including sepsis, septic shock or sepsis-like conditions and IBD.

This application is a continuation of U.S. patent application Ser. No.12/320,707, filed Feb. 2, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 11/284,086, filed on Nov. 22, 2005, whichclaims priority from United Kingdom Patent Application No. 0426146.7,filed on Nov. 29, 2004, and Japanese Patent Application No. 2005-146848,filed on May 19, 2005. The contents of these applications areincorporated herein by reference in their entirety.

The present invention relates to the field of immunology. Moreparticularly, the present invention relates to inflammation and the useof proteins and peptides containing certain sequences of the TREM-1protein and their functional equivalents (referred to herein asTREM1-peptides) in the treatment of disease, for example, sepsis, septicshock and inflammatory bowel disease (IBD).

Sepsis constitutes a significant consumption of intensive care resourcesand remains an ever-present problem in the intensive care unit. It hasbeen estimated that between 400 000 and 500 000 patients are so affectedeach year in both the USA and Europe. Morbidity and mortality haveremained high despite improvements in both supportive and anti-microbialtherapies. Mortality rates vary from 40% for uncomplicated sepsis to 80%in those suffering from septic shock and multi-organ dysfunction. Thepathogenesis of the conditions is now becoming better understood.Greater understanding of the complex network of immune, inflammatory andhaematological mediators may allow the development of rational and noveltherapies.

Following an infection, innate and cognitive immune responses develop insequential phases that build-up in specificity and complexity, resultingultimately in the clearance of infectious agents and restoration ofhomeostasis. The innate immune response serves as the first line ofdefence and is initiated upon activation of pattern recognitionreceptors, such as Toll-like receptors (TLRs) (1, 2), by variouspathogen-associated microbial patterns (PAMPs) (3). Activation of theTLRs triggers the release of large quantities of such cytokines as TNF-αand IL-1β, which, in case of such massive infections as sepsis, canprecipitate tissue injury and lethal shock (4, 5). Although antagonistsof TNF-α and IL-1β appeared in this context as possibly interestingtherapeutic agents of sepsis, they have unfortunately shown limitedefficacy in clinical trials (6-8). This could be due to the fact thatthese cytokines are necessary for the clearance of infections, and thattheir removal would allow for fatal bacterial growth (9-11).

Another receptor involved in, inter alia, response to infection,triggering receptor expressed on myeloid cells-1 (TREM-1) is a member ofa recently discovered family of receptors, the TREM family, expressed onthe surface of neutrophils and a subset of monocytes. TREM receptorsactivate myeloid cells via association with the adaptor molecule DAP12.Engagement of TREM-1 has been reported to trigger the synthesis ofpro-inflammatory cytokines in the presence of microbial products.

The triggering receptor expressed on myeloid cells (TREM)-1 is arecently discovered cell-surface molecule that has been identified bothon human and murine polymorphonuclear neutrophils and mature monocytes(12). It belongs to the immunoglobulin superfamily and activatesdownstream signalling pathways with the help of an adapter proteincalled DAP12 (12-15). Bouchon and co-workers have shown that theexpression of TREM-1 was greatly up-regulated on neutrophils andmonocytes in the presence of such bacteria as Pseudomonas aeruginosa orStaphylococcus aureus, both in cell culture and in tissue samples frompatients with infection (16). In striking contrast, TREM-1 was notup-regulated in samples from patients with non-infectious inflammatorydiseases such as psoriasis, ulcerative colitis or vasculitis caused byimmune complexes (16). Moreover, when TREM-1 is bound to its ligand,there is a synergistic effect of LPS and an amplified synthesis of thepro-inflammatory cytokines TNF-α and GM-CSF, together with an inhibitionof IL-10 production (17). In a murine model of LPS-induced septic shock,blockade of TREM-1 signalling protected the animals from death, furtherhighlighting the crucial role of this molecule (13, 16).

Recent studies demonstrate that TREM-1 plays a critical role in theinflammatory response to infection (see BOUCHON et al. (2000) J.Immunol. 164:4991-4995). Expression of TREM-1 is increased on myeloidcells in response to both bacterial and fungal infections in humans.Similarly, in mice the induction of shock by lipopolysaccharide (LPS) isassociated with increased expression of TREM-1. Further, treatment ofmice with a soluble TREM-1/Ig fusion protein, as a ‘decoy’ receptor,protects mice from death due to LPS or E.coli.

Triggering via TREM-1 results in the production of pro-inflammatorycytokines, chemokines and reactive oxygen species, and leads to rapiddegranulation of neutrophilic granules, and phagocytosis. Sinceinterfering with TREM-1 engagement leads to the simultaneous reductionin production and secretion of a variety of proinflammatory mediators,TREM-1 represents an attractive target for treating chronic inflammatorydisorders. Indeed, a role for TREM-1 has been demonstrated in a varietyof inflammatory disorders, including (but not limited to) acute andchronic inflammatory disorders, sepsis, acute endotoxemia, encephalitis,Chronic Obstructive Pulmonary Disease (COPD), allergic inflammatorydisorders, asthma, pulmonary fibrosis, pneumonia, Community acquiredpneumonia (CAP), Ventilator associated pneumonia (VAP), Acuterespiratory infection, Acute respiratory distress syndrome (ARDS),Infectious lung diseases, Pleural effusion, Peptic ulcer, Helicobacterpylori infection, hepatic granulomatosis, arthritis, rheumatoidarthritis, osteoarthritis, inflammatory osteolysis, ulcerative colitis,psoriasis, vasculitis, autoimmune disorders, thyroiditis, Meliodosis,(mesenteric) Ischemia reperfusion, Filovirus infection, Infection of theurinary tract, Bacterial meningitis, Salmonella enterica infection,Marburg and Ebola viruses infections, and in particular InflammatoryBowel Disease (IBD). Inflammatory bowel disease (IBD) covers a group ofdisorders in which the intestines become inflamed (red and swollen),probably as a result of an immune reaction of the body against its ownintestinal tissue. Two major types of IBD are described: ulcerativecolitis (UC) and Crohn disease (CD). Ulcerative colitis is limited tothe colon (large intestine). Crohn disease can involve any part of thegastrointestinal tract from the mouth to the anus, but it most commonlyaffects the small intestine and/or the colon. Both ulcerative colitisand Crohn disease vary in the intensity and severity during the courseof the disease. When there is severe inflammation, the disease isconsidered to be in an active stage, and the person experiences aflare-up of the condition. When the degree of inflammation is less (orabsent), the person usually is without symptoms, and the disease isconsidered to be in remission. In IBD factor or factors trigger thebody's immune system to produce an inflammatory reaction in theintestinal tract that continues without control. As a result of theinflammatory reaction, the intestinal wall is damaged leading to bloodydiarrhea and abdominal pain.

U.S. Pat. No. 6,420,526 entitled “186 Secreted Proteins” claimsunspecified and unexemplified isolated fragments of TREM-1 containing atleast 30 contiguous amino acids of human TREM-1. No biological datarelating to such fragments are provided.

As described in US2003165875A, fusion proteins between human IgG1constant region and the extracellular domain of mouse TREM-1 or that ofhuman TREM-1 show an effect against endotoxemia in mice.

The inventors have surprisingly found that certain peptides derived fromthe TREM-1 protein are capable of acting as antagonists of the TREM-1protein and therefore have applications in the treatment of sepsis,septic shock and inflammatory bowel disease (IBD). The Inventors furtherdemonstrate that the same peptides also modulate in vivo thepro-inflammatory cascade triggered by infection, thus inhibitinghyper-responsiveness and death in an animal model of sepsis, and thatblocking TREM-1 attenuates the symptoms of IBD in mice

Previously, the Inventors have identified a soluble form of TREM-1(sTREM-1) and observed significant levels in serum samples from septicshock patients but not controls. As also described herein the Inventorshave investigated its putative role in the modulation of inflammationduring sepsis (see Gibot et al. (2004) Ann. Intern. Med. 141(1):9-15 andGibot et al. (2004) N. Engl. J. Med. 350(5):451-8).

As described herein the Inventors show that a soluble form of TREM-1(sTREM-1) is released in the peripheral blood during infectiousaggression in mouse. The Inventors also confirm monocytes as a majorsource of sTREM, and show that synthetic peptides mimicking a part ofthe extra-cellular domain of TREM-1 can modulate cytokine production byactivated monocytes in vitro.

The Inventors have observed that sTREM-1 is secreted by monocytesactivated in vitro by LPS, as well as in the serum of animals involvedin an experimental model of septic shock. Both in vitro and in vivo,synthetic peptides mimicking a short highly conserved domain of sTREM-1attenuate cytokine production by human monocytes and protect septicanimals from hyper-responsiveness and death. These peptides areefficient not only in preventing but also in down-regulating thedeleterious effects of pro-inflammatory cytokines. These datademonstrate that in vivo modulation of TREM-1 by TREM-1 peptides is avaluable therapeutic tool for the treatment of infection, for examplesepsis or septic shock or for the treatment of sepsis-like conditions

Accordingly, the present invention provides methods and compositions forthe treatment of infectious disease, in particular, sepsis and septicshock or for the treatment of sepsis-like conditions

Other diseases or disorders that may also be treated by the methods andcompositions of the present invention include any inflammatory disorder(or other disorder) that is mediated by the binding of the TREM-1 ligandto a TREM-1 receptor. Examples of inflammatory disorders include (butare not limited to) acute and chronic inflammatory disorders, sepsis,acute endotoxemia, encephalitis, Chronic Obstructive Pulmonary Disease(COPD), allergic inflammatory disorders, asthma, pulmonary fibrosis,pneumonia, Community acquired pneumonia (CAP), Ventilator associatedpneumonia (VAP), Acute respiratory infection, Acute respiratory distresssyndrome (ARDS), Infectious lung diseases, Pleural effusion, Pepticulcer, Helicobacter pylori infection, hepatic granulomatosis, arthritis,rheumatoid arthritis, osteoarthritis, inflammatory osteolysis,ulcerative colitis, psoriasis, vasculitis, autoimmune disorders,thyroiditis, Meliodosis, (mesenteric) lschemia reperfusion, Filovirusinfection, Infection of the urinary tract, Bacterial meningitis,Salmonella enterica infection, Marburg and Ebola viruses infections, andin particular Inflammatory Bowel Disease (IBD),

As described herein, the Inventors have determined that several peptidesof the extracellular portion of the TREM-1 protein (see Table 1), whichincorporate sequences from “CDR2” and “CDR3” surprisingly have activitysimilar to previously described fusion proteins of IgG1 constant regionand the extracellular domain of TREM-1 in models of sepsis. Thesepeptides also have advantages over the protein particularly in terms ofcost of manufacture.

Thus, the invention provides polypeptides comprising one or moresequences derived from CDR2 or CDR3 of a TREM-1 protein. Preferably,said polypeptides comprise less than 30 contiguous amino acids of saidTREM-1 protein.

As shown in Table 1, examples of such peptides or polypeptides, containor comprise for example 15-25 amino acid (“AA”) peptides from the TREM-1protein and contain or comprise all or part of a CDR domain (3-6 AAs) ofthe receptor flanked by natural sequences from the protein that can varyin length so long as function of the CDR-like domain is not lost. Suchpeptides are derived from the TREM-1 receptor protein amino acidsequence for example, as shown in Table 2 (human) and Table 3 (mouse).

Table 1 shows peptides derived from mouse TREM-1 “mPX” (NCBI ReferenceSequences (RefSeq) NP_(—)067381) or human TREM-1 “hPX″” (NCBI ReferenceSequences (RefSeq) NP_(—)061113). Underlined amino acids span the humanTREM-1 Complementarity Determining Regions (CDR), as described by Radaevet al. 2003 Structure (Camb.) 11 (12), 1527-1535 (2003).

Table 2 shows the human TREM-1 amino acid sequence NP_(—)061113.Underlined amino acids span the human TREM-1 Complementarity DeterminingRegions (CDR) 2 (RPSKNS; [SEQ ID NO:20]) and 3 (QPPKE [SEQ ID NO:21]),as described by Radaev et al. 2003 Structure (Camb.) 11 (12), 1527-1535(2003).

Table 3 shows the mouse TREM-1 amino acid sequence NP_(—)067381.Underlined amino acids span the mouse TREM-1 Complementarity DeterminingRegions (CDR) 2 (RPFTRP; [SEQ ID NO:22]) and 3 (HPPND; [SEQ ID NO:23]).

TABLE 1 Peptides including sequences from human and mouseTREM-1 CDR 2 and CDR 3 hCDR 2 mP1 (67-89): [SEQ ID NO: 3]LVVTQRPFTRPSEVHMGKFTLKH hP1 (67-89): [SEQ ID NO: 16]LACTERPSKNSHPVQVGRIILED hCDR 3 mP2 (114-136): [SEQ ID NO: 4]           VIYHPPNDPVVLFHPVRLVVTKG mP4 (103-123): [SEQ ID NO: 6]LQVTDSGLYRCVIYHPPNDPV mP5 (103-119): [SEQ ID NO: 7] LQVTDSGLYRCVIYHPPhP2 (114-136): [SEQ ID NO: 17]            VIYQPPKEPHMLFDRIRLVVTKGhP4 (103-123): [SEQ ID NO: 18] LQVEDSGLYQCVIYQPPKEPH hP5 (103-119):[SEQ ID NO: 19] LQVEDSGLYQCVIYQPP

TABLE 2 Human TREM-1 amino acid sequence NP_061113 1MRKTRLWGLL WMLFVSELRA ATKLTEEKYE LKEGQTLDVK CDYTLEKFAS SQKAWQIIRD 61GEMPKTLACT ERPSKNSHPV QVGRIILEDY HDHGLLRVRM VNLQVEDSGL YQCVIYQPPK 121EPHMLFDRIR LVVTKGFSGT PGSNENSTQN VYKIPPTTTK ALCPLYTSPR TVTQAPPKST 181ADVSTPDSEI NLTNVTDIIR VPVFNIVILL AGGFLSKSLV FSVLFAVTLR SFVP[SEQ ID NO: 1]

TABLE 3 Mouse TREM-1 amino acid sequence NP_067381 1MRKAGLWGLL CVFFVSEVKA AIVLEEERYD LVEGQTLTVK CPFNIMKYAN SQKAWQRLPD 61GKEPLTLVVT QRPFTRPSEV HMGKFTLKHD PSEAMLQVQM TDLQVTDSGL YRCVIYHPPN 121DPVVLFHPVR LVVTKGSSDV FTPVIIPITR LTERPILITT KYSPSDTTTT RSLPKPTAVV 181SSPGLGVTII NGTDADSVST SSVTISVICG LLSKSLVFII LFIVTKRTFG [SEQ ID NO: 2]

Accordingly, the invention provides isolated or recombinantly preparedpolypeptides or peptides comprising or consisting essentially of one ormore sequences derived from CDR2 or CDR3 of a TREM-1 protein, orfragments, homologues, derivatives, fusion proteins or variants of suchpolypeptides, as defined herein, which are herein collectively referredto as “polypeptides or peptides of the invention” or “TREM-1 peptides orTREM-1 polypeptides”, preferably such entities comprise less than 30contiguous amino acids of a TREM-1 protein, for example as shown inTable 2 or Table 3. Generally where polypeptides or proteins of theinvention or fragments, homologues, derivatives, or variants thereof areintended for use (for example treatment) in a particular species, thesequences of CDR2 or CDR3 of a TREM-1 protein are chosen from the TREM-1protein amino acid sequence of that species, or if the sequence is notknown, an analogous species. For example, polypeptides or proteins ofthe invention for the treatment of human disease, in particular sepsis,septic shock or sepsis-like conditions, will comprise one or moresequences comprising all or part of CDR2 or CDR3 from the human TREM-1protein.

Furthermore, the invention provides isolated polypeptides or proteinscomprising an amino acid sequence that is at least about 60%, 70%, 75%,80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQID NO:20, 21, 22, 23 or fragments, homologues, derivatives, or variantsthereof. The invention also provides isolated peptides, polypeptides orproteins comprising an amino acid sequence that comprises or consists ofat least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21 22, 23, 24, 25, 26, 27, 28, or 29 or more contiguous aminoacids of a TREM-1 protein of which 3 or more contiguous amino acids arederived from the sequences of SEQ ID NO:20, 21, 22 or 23 (in other wordsa sequence representing all, or part of CDR2 or CDR3 of a TREM-1 proteinis present in the peptide, polypeptide or protein), or fragments,homologues, derivatives, or variants thereof. In preferred embodiments,such peptides, polypeptides or proteins, or fragments, homologues,derivatives or variants thereof have a biological activity of a TREM-1full-length protein, such as antigenicity, immunogenicity, triggering ofproinflammatory chemokines and cytokines, mobilization of cytosolicCa²⁺, protein tyrosine-phosphorylation, mediator release, and otheractivities readily assayable. Generally, such peptides, polypeptides orproteins or fragments, homologues, derivatives or variants thereof arecapable of treating sepsis, septic shock or sepsis-like conditions, orare active in experimental models of sepsis, septic shock or sepsis-likeconditions, for example by acting as antagonists of the activity of theTREM-1 receptor. Such peptides, polypeptides or proteins or fragments,homologues, derivatives or variants thereof are characterised by theability to treat, ameliorate, or lessen the symptoms of sepsis, septicshock or sepsis-like conditions.

In particular, the invention provides, a TREM-1 polypeptide havingactivity against sepsis, septic shock or sepsis-like conditions whichconsists of (i) a contiguous sequence of 5 to 29, for example 15-25,amino acids corresponding to the native TREM-1 protein sequence whichincludes at least 3 amino acids from the CDR2 or CDR3 sequences; or (ii)such a sequence in which one or more (e.g. one, two or three) aminoacids are substituted conservatively with another amino acid provided,however that at least 3 amino acids from the CDR2 or CDR3 sequences arenot substituted; or (iii) a sequence of (i) or (ii) linked at one orboth of its N and C termini to a heterologous polypeptide. For example,in a polypeptide wherein the native TREM-1 protein sequence is the humansequence identified as (SEQ ID NO: 1), the CDR2 and CDR3 sequences areRPSKNS (SEQ ID NO:20) and QPPKE (SEQ ID NO:21) respectively. In suchpolypeptides, the at least 3 amino acids from the CDR2 or CDR3 sequencescan be QPP, PPK, PKE, RPS, PSK, SKN or KNS. Such polypeptides maycomprise the sequence QPPK ([SEQ ID NO:24), QPPKE (SEQ ID NO:21) orRPSKNS (SEQ ID NO:20). For example, in a polypeptide wherein the nativeTREM-1 protein sequence is the mouse sequence identified as (SEQ ID NO:2) the CDR2 and CDR3 sequences are RPFTRP (SEQ ID NO:22) and HPPND (SEQID NO:23) respectively. In such polypeptides, the at least 3 amino acidsfrom the CDR2 or CDR3 sequences can be HPP, PPN, PND, RPF, PFT, FTR orTRP. Such polypeptides may comprise the sequences HPP, HPPN (SEQ IDNO:25), HPPND (SEQ ID NO:23) or RPFTRP (SEQ ID NO:22).

In certain embodiments, the polypeptide of the invention is or comprisesSEQ ID No. 7 which is disclosed in Gibot et al (2004) J Exp Med 200,1419-1426.

In certain embodiments the polypeptide of the invention neither is norcomprises SEQ ID No. 7.

In certain embodiments the polypeptide of the invention is or comprisesa sequence selected from SEQ ID Nos. 3, 4 and 6.

In certain embodiments the polypeptide of the invention is or comprisesa sequence selected from SEQ ID Nos. 16, 17, 18 and 19.

In certain embodiments the polypeptide of the invention is or comprisesa sequence derived from CDR2.

In certain embodiments the polypeptide of the invention is or comprisesa sequence derived from CDR3.

The polypeptides or peptides of the invention are provided for use intherapy, in particular in the treatment of sepsis, septic shock andsepsis-like conditions, and for use in the manufacture of a medicamentfor the treatment of sepsis, septic shock and sepsis-like conditions.Further provided are compositions and pharmaceutical compositionscontaining polypeptides or peptides of the invention and methods oftreatment of sepsis, septic shock and sepsis-like conditions usingpolypeptides or peptides of the invention. In addition the polypeptidesor peptides of the invention are provided for use in therapy to restorehaemodynamic parameters in sepsis, septic shock and sepsis-likeconditions and for use in the manufacture of a medicament for thetreatment of aberrant haemodynamic parameters in sepsis, septic shockand sepsis-like conditions.

The term “triggering receptor expressed on myeloid cells” or “TREM”refers to a group of activating receptors which are selectivelyexpressed on different types of myeloid cells, such as mast cells,monocytes, macrophages, dendritic cells (DCs), and neutrophils, and mayhave a predominant role in immune and inflammatory responses. TREMs areprimarily transmembrane glycoproteins with a Ig-type fold in theirextracellular domain and, hence, belong to the Ig-SF. These receptorscontain a short intracellular domain, but lack docking motifs forsignaling mediators and require adapter proteins, such as DAP12, forcell activation.

The term “myeloid cells” as used herein refers to a series of bonemarrow-derived cell lineages including granulocytes (neutrophils,eosinophils, and basophils), monocytes, macrophages, and mast cells.Furthermore, peripheral blood dendritic cells of myeloid origin, anddendritic cells and macrophages derived in vitro from monocytes in thepresence of appropriate culture conditions, are also included.

The term “sepsis, septic shock” or “sepsis or septic shock” as definedherein, refers to sub-groups of systemic inflammatory response syndrome(SIRS). The term “sepsis” is generally reserved for SIRS when infectionis suspected or proven. A pattern of physiological variables have beenshown in critically ill patients in response to a range of insultsincluding; trauma, burns, pancreatitis and infection. These includeinflammatory responses, leucocytosis or severe leucopaenia, hyperthermiaor hypothermia, tachycardia and tachypnoea and have been collectivelytermed the systemic inflammatory response syndrome (SIRS). Thisdefinition emphasises the importance of the inflammatory process inthese conditions regardless of the presence of infection. Sepsis isfurther stratified into severe sepsis when there is evidence of organhypoperfusion, made evident by signs of organ dysfunction such ashypoxaemia, oliguria, lactic acidosis or altered cerebral function.“Septic shock” is severe sepsis usually complicated by hypotension,defined in humans as systolic blood pressure less than 90 mmHg despiteadequate fluid resuscitation. Sepsis and SIRS may be complicated by thefailure of two or more organs, termed multiple organ failure (MOF), dueto disordered organ perfusion and oxygenation. In addition to systemiceffects of infection, a systemic inflammatory response may occur insevere inflammatory conditions such as pancreatitis and burns. Theappearance of signs of an inflammatory response is less well definedfollowing traumatic insults. In the intensive care unit, gram-negativebacteria are implicated in 50 to 60% of sepsis cases with gram-positivebacteria accounting for a further 35 to 40% of cases. The remainder ofcases are due to the less common causes of fungi, viruses and protozoa.

The term “sepsis-like conditions” as used herein refers to those statesin which a patient presents with symptoms similar to sepsis or septicshock but where an infectious agent is not the primary or initial causeof a similar cascade of inflammatory mediators and/or change inhaemodynamic parameters as seen in cases of sepsis, for example inpatients with acute or chronic liver failure (see Wasmuth H E, et al. JHepatol. 2005 February; 42(2):195-201), in cases of post-resuscitationdisease after cardiac arrest (see Adrie C et al. Curr Opin Crit Care.2004 June; 10(3):208-12) in the treatment of sepsis-like symptoms aftercancer chemotherapy (see Tsuji E et al. Int J Cancer. 2003 Nov. 1;107(2):303-8) in patients undergoing hyperthermic isolated limbperfusion with recombinant TNF-alpha or similar treatments (seeZwaveling J H et al. Crit Care Med. 1996 May; 24(5):765-70) orsepsis-like illness in neonates (see Griffin M P et al. Pediatr Res.2003 June; 53(6):920-6).

The term “activity against sepsis, septic shock or sepsis-likeconditions” as used herein refers to the capability of a molecule, forexample a peptide, polypeptide or engineered antibody, to treat sepsis,septic shock or sepsis-like conditions, or be active in experimentalmodels of sepsis, septic shock or sepsis-like conditions, for example byacting as an antagonist of the activity of the TREM-1 receptor.

Typically the indication for polypeptides of the invention is sepsis orseptic-shock or Inflammatory Bowel Disease (IBD). Other indications mayinclude any inflammatory disorder (or other disorder) that is mediatedby the binding of the TREM-1 ligand to a TREM-1 receptor. Examples ofinflammatory disorders include (but are not limited to) acute andchronic inflammatory disorders, sepsis, acute endotoxemia, encephalitis,Chronic Obstructive Pulmonary Disease (COPD), allergic inflammatorydisorders, asthma, pulmonary fibrosis, pneumonia, Community acquiredpneumonia (CAP), Ventilator associated pneumonia (VAP), Acuterespiratory infection, Acute respiratory distress syndrome (ARDS),Infectious lung diseases, Pleural effusion, Peptic ulcer, Helicobacterpylon infection, hepatic granulomatosis, arthritis, rheumatoidarthritis, osteoarthritis, inflammatory osteolysis, ulcerative colitis,psoriasis, vasculitis, autoimmune disorders, thyroiditis, Meliodosis,(mesenteric) lschemia reperfusion, Filovirus infection, Infection of theurinary tract, Bacterial meningitis, Salmonella enterica infection,Marburg and Ebola viruses infections.

The term “substantial sequence identity”, when used in connection withpeptides/amino acid sequences, refers to peptides/amino acid sequenceswhich are substantially identical to or similar in sequence, giving riseto a homology in conformation and thus to similar biological activity.The term is not intended to imply a common evolution of the sequences.

Typically, peptides/amino acid sequences having “substantial sequenceidentity” are sequences that are at least 50%, more preferably at least80%, identical in sequence, at least over any regions known to beinvolved in the desired activity. Most preferably, no more than fiveresidues, other than at the termini, are different. Preferably, thedivergence in sequence, at least in the aforementioned regions, is inthe form of “conservative modifications”

To determine the percent sequence identity of two peptides/amino acidsequences or of two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in one orboth of a first and a second amino acid or nucleic acid sequence foroptimal alignment and non-homologous sequences can be disregarded forcomparison purposes). For example, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence (e.g., when aligning a second sequence to the firstamino acid sequence which has for example 100 amino acid residues, atleast 30, preferably at least 40, more preferably at least 50, even morepreferably at least 60, and even more preferably at least 70, 80 or 90amino acid residues are aligned). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch (J. Mol.Biol. (48):444-453 (1970)) algorithm which has been incorporated intothe GAP program in the GCG software package (available online at the GCGwebsite), using either a Blossom 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4,5, or 6. In another embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available online at the GCG website), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80, and alength weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percentidentity between two amino acid or nucleotide sequences is determinedusing the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989))which has been incorporated into the ALIGN program (version 2.0), usinga PAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4. The nucleic acid and protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to identify, for example, other family membersor related sequences. Such searches can be performed using the NBLASTand XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to NIP2b, NIP2cL, and NIP2cS nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to NIP2b, NIP2cL, and NIP2cS protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. These programs are publically availableon the National Center for Biotechnology Information (“NCBI”) website.

The terms “protein” and “polypeptide” are used interchangeably herein.The term “peptide” is used herein to refer to a chain of two or moreamino acids or amino acid analogues (including non-naturally occurringamino acids), with adjacent amino acids joined by peptide (—NHCO—)bonds. Thus, the peptides of the invention include oligopeptides,polypeptides, proteins, mimetopes and peptidomimetics. Methods forpreparing mimetopes and peptidomimetics are known in the art.

The terms “mimetope” and “peptidomimetic” are used interchangeablyherein. A “mimetope” of a compound X refers to a compound in whichchemical structures of X necessary for functional activity of X havebeen replaced with other chemical structures which mimic theconformation of X. Examples of peptidomimetics include peptidiccompounds in which the peptide backbone is substituted with one or morebenzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science260:1937-1942) and “retro-inverso” peptides (see U.S. Pat. No. 4,522,752to Sisto). The terms “mimetope” and “peptidomimetic” also refer to amoiety, other than a naturally occurring amino acid, thatconformationally and functionally serves as a substitute for aparticular amino acid in a peptide-containing compound without adverselyinterfering to a significant extent with the function of the peptide.Examples of amino acid mimetics include D-amino acids. Peptidessubstituted with one or more D-amino acids may be made using well knownpeptide synthesis procedures. Additional substitutions include aminoacid analogues having variant side chains with functional groups, forexample, b-cyanoalanine, canavanine, djenkolic acid, norleucine,3-phosphoserine, homoserine, dihydroxyphenylalanine,5-hydroxytryptophan, 1-methylhistidine, or 3-methylhistidine

As used herein an “analogue” of a compound X refers to a compound whichretains chemical structures of X necessary for functional activity of X,yet which also contains certain chemical structures which differ from X.An example of an analogue of a naturally-occurring peptide is a peptidewhich includes one or more non-naturally-occurring amino acids. The term“analogue” is also intended to include modified mimetopes and/orpeptidomimetics, modified peptides and polypeptides, and allelicvariants of peptides and polypeptides. Analogues of a peptide willtherefore produce a peptide analogue that is substantially homologousor, in other words, has substantial sequence identity to the originalpeptide. The term “amino acid” includes its art recognized meaning andbroadly encompasses compounds of formula I:

Preferred amino acids include the naturally occurring amino acids, aswell as synthetic derivatives, and amino acids derived from proteins,e.g., proteins such as casein, i.e., casamino acids, or enzymatic orchemical digests of, e.g., yeast, an animal product, e.g., a meatdigest, or a plant product, e.g., soy protein, cottonseed protein, or acorn steep liquor (see, e.g., Traders' Guide to Fermentation Media,Traders Protein, Memphis, Tenn. (1988), Biotechnology: A Textbook ofIndustrial Microbiology, Sinauer Associates, Sunderland, Mass. (1989),and Product Data Sheet for Corn Steep Liquor, Grain Processing Corp.,IO).

The term “naturally occurring amino acid” includes any of the 20 aminoacid residues which commonly comprise most polypeptides in livingsystems, rarer amino acids found in fibrous proteins (e.g.,4-hydorxyproline, 5-hydroxylysine, —N-methyllysine, 3-methylhistidine,desmosine, isodesmosine), and naturally occurring amino acids not foundin proteins (e.g., -aminobutryic acid, homocysteine, homoserine,citrulline, ornithine, canavanine, djenkolic acid, and -cyanoalanine).

The term “side chain of a naturally occurring amino acid” is intended toinclude the side chain of any of the naturally occurring amino acids, asrepresented by R in formula I. One skilled in the art will understandthat the structure of formula I is intended to encompass amino acidssuch as proline where the side chain is a cyclic or heterocyclicstructure (e.g., in proline R group and the amino group form afive-membered heterocyclic ring.

The term “homologue,” as used herein refers to any member of a series ofpeptides or polypeptides having a common biological activity, includingantigenicity/immunogenicity and inflammation regulatory activity, and/orstructural domain and having sufficient amino acid as defined herein.Such homologues can be from either the same or different species ofanimals.

The term “variant” as used herein refers either to a naturally occurringallelic variation of a given peptide or a recombinantly preparedvariation of a given peptide or protein in which one or more (e.g. one,two or three) amino acid residues have been modified by amino acidsubstitution, addition, or deletion.

The term “derivative” as used herein refers to a variation of givenpeptide or protein that are otherwise modified, i.e., by covalentattachment of any type of molecule, preferably having bioactivity, tothe peptide or protein, including non-naturally occurring amino acids.

Preferably, such homologues, variants and derivatives are capable oftreating sepsis, septic shock or sepsis-like conditions, or are activein experimental models of sepsis, septic shock or sepsis-likeconditions, or are capable of treating IBD or other inflammatorydisorder, for example by acting as antagonists of the activity of theTREM-1 receptor.

An “isolated” or “purified” peptide or protein is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.

The language “substantially free of cellular material” includespreparations of a polypeptide/protein in which the polypeptide/proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a polypeptide/protein that issubstantially free of cellular material includes preparations of thepolypeptide/protein having less than about 30%, 20%, 10%, 5%, 2.5%, or1%, (by dry weight) of contaminating protein. When thepolypeptide/protein is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When polypeptide/protein is produced by chemical synthesis,it is preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the polypeptide/protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than polypeptide/protein fragment of interest. In apreferred embodiment of the present invention, polypeptides/proteins areisolated or purified.

In addition to the polypeptides described above, polypeptides of theinvention also encompass those polypeptides having a common biologicalactivity and/or structural domain and having sufficient amino acididentity (homologues) as defined herein. These homologues can be fromeither the same or different species of animal, preferably from mammals,more preferably from rodents, such as mouse and rat, and most preferablyfrom human. Preferably, they exhibit at least one structural and/orfunctional feature of TREM-1, and are preferably, capable of treatingsepsis, septic shock or sepsis-like conditions, for example by acting asantagonists of the activity of the TREM-1 receptor. Such modificationsinclude amino acid substitution, deletion, and/or insertion. Amino acidmodifications can be made by any method known in the art and variousmethods are available to and routine for those skilled in the art.

Additionally, in making amino acid substitutions, generally the aminoacid residue to be substituted can be a conservative amino acidsubstitution (i.e. “substituted conservatively”), for example, a polarresidue is substituted with a polar residue, a hydrophilic residue witha hydrophilic residue, hydrophobic residue with a hydrophobic residue, apositively charged residue with a positively charged residue, or anegatively charged residue with a negatively charged residue. Moreover,generally, the amino acid residue to be modified is not highly orcompletely conserved across species and/or is critical to maintain thebiological activities of the peptide and/or the protein it derives from.

Peptides of the invention may be directly synthesised in any convenientway. Generally the reactive groups present (for example amino, thioland/or carboxyl) will be protected during overall synthesis. Aproportion of the peptides of the invention, i.e. those wherein thecomprised amino acids are genetically coded amino acids, will be capableof being expressed in prokaryotic and eukaryotic hosts by expressionsystems well known to the man skilled in the art. Methods for theisolation and purification of e. g. microbially expressed peptides arealso well known. Polynucleotides which encode these peptides of theinvention constitute further aspects of the present invention. As usedherein, “polynucleotide” refers to a polymer of deoxyribonucleotides orribonucleotides, in the form of a separate fragment or as a component ofa larger construct, e. g. an expression vector such as a plasmid.Polynucleotide sequences of the invention include DNA, RNA and cDNAsequences. Due to the degeneracy of the genetic code, of course morethan one polynucleotide is capable of encoding a particular peptideaccording to the invention. When a bacterial host is chosen forexpression of a peptide, it may be necessary to take steps to protectthe host from the expressed anti-bacterial peptide. Such techniques areknown in the art and include the use of a bacterial strain which isresistant to the particular peptide being expressed or the expression ofa fusion peptide with sections at one or both ends which disable theantibiotic activity of the peptide according to the invention. In thelatter case, the peptide can be cleaved after harvesting to produce theactive peptide. If the peptide incorporates a chemical modification thenthe activity/stability of the expressed peptide may be low, and is onlymodulated by post-synthetic chemical modification

Furthermore, the invention also encompasses derivatives of thepolypeptides of the invention. For example, but not by way oflimitation, derivatives may include peptides or proteins that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativemay contain one or more non-classical amino acids. Those skilled in theart will be aware of various methods for modifying peptides to increasepotency, prolong activity and/or increase half-life. In one example(WO0210195) the modification is made via coupling through an amide bondwith at least one conformationally rigid substituent, either at theN-terminal of the peptide, the C-terminal of the peptide, or on a freeamino or carboxyl group along the peptide chain. Other examples ofpeptide modifications with similar effects are described, for example,in WO2004029081, WO03086444, WO03049684, WO0145746, WO0103723 andWO9101743.

The invention further provides antibodies that comprise a peptide orpolypeptide of the invention or that mimic the activity of peptides orpolypeptides of the invention. Such antibodies include, but are notlimited to: polyclonal, monoclonal, bi-specific, multi-specific, human,humanized, chimeric antibodies, single chain antibodies, Fab fragments,F(ab′)2 fragments, disulfide-linked Fvs, and fragments containing eithera VL or VH domain or even a complementary determining region (CDR) thatspecifically binds to a polypeptide of the invention. In anotherembodiment, antibodies can also be generated using various phage displaymethods known in the art. Techniques to recombinantly produce Fab, Fab′and F(ab′)2 fragments can also be employed using methods known in theart such as those disclosed in PCT publication WO 92/22324; Mullinax, etal., BioTechniques, 12(6):864-869, 1992; and Sawai, et al., 1995, AJRI34:26-34; and Better, et al., 1988, Science 240:1041-1043 (each of whichis incorporated by reference in its entirety). Examples of techniquesthat can be used to produce single-chain Fvs and antibodies includethose described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston, etal., 1991, Methods in Enzymology 203:46-88; Shu, et al., 1993, Proc.Natl. Acad. Sci. USA 90:7995-7999; and Skerra, et al., 1988, Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a constant region derivedfrom a human immunoglobulin. Methods for producing chimeric antibodiesare known in the art. See, e.g., Morrison, 1985, Science 229:1202; Oi,et al., 1986, BioTechniques 4:214; Gillies, et al., 1989, J. Immunol.Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397;which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule or in the case of the present invention,one or more CDRs derived from a TREM-1 protein. As known in the art,framework residues in the human framework regions can be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modelling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. See, e.g.,Queen, et al., U.S. Pat. No. 5,585,089; Riechmann, et al., 1988, Nature332:323, 1988, which are incorporated herein by reference in theirentireties. Antibodies can be humanized using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,1991, Molecular Immunology, 28(4/5):489-498; Studnicka, et al., 1994,Protein Engineering, 7(6):805-814; Roguska, et al., 1994, Proc Natl.Acad. Sci. USA 91:969-973, and chain shuffling (U.S. Pat. No.5,565,332), all of which are hereby incorporated by reference in theirentireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety. Human antibodies can also beproduced using transgenic mice (see Lonberg and Huszar (1995), Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; which areincorporated by reference herein in their entireties. In addition,companies such as Abgenix, Inc. (Freemont, Calif.), Medarex (N.J.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above. Completely human antibodies which recognize a selectedepitope can be generated using a technique referred to as “guidedselection.” In this approach a selected non-human monoclonal antibody,e.g., a mouse antibody, is used to guide the selection of a completelyhuman antibody recognizing the same epitope. (Jespers et al., 1988,Bio/technology 12:899-903). Antibodies fused or conjugated toheterologous polypeptides may be used in in vitro immunoassays and inpurification methods (e.g., affinity chromatography) well known in theart. See, e.g., PCT publication Number WO 93/21232; EP 439,095;Naramura, et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No.5,474,981; Gillies, et al., 1992 Proc. Natl. Acad. Sci. USA89:1428-1432; and Fell, et al., 1991, J. Immunol. 146:2446-2452, whichare incorporated herein by reference in their entireties.

In another aspect, the present invention provides methods foridentifying a compound or ligand that binds to or modulates the activityof a polypeptide of the invention. Such a method comprises measuring abiological activity of the polypeptide in the presence or absence of atest compound and identifying test compounds that alter (increase ordecrease) the biological activity of the polypeptide.

In one embodiment, the invention provides a fusion protein comprising abioactive molecule and one or more domains of a polypeptide of theinvention or fragment thereof. In particular, the present inventionprovides fusion proteins comprising a bioactive molecule recombinantlyfused or chemically conjugated (including both covalent and non-covalentconjugations) to one or more domains of a polypeptide of the inventionor fragments thereof.

The present invention further encompasses fusion proteins in which thepolypeptides of the invention or fragments thereof, are recombinantlyfused or chemically conjugated (including both covalent and non-covalentconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences.

In one example, a fusion protein in which a polypeptide of the inventionor a fragment thereof can be fused to sequences derived from varioustypes of immunoglobulins. For example, a polypeptide of the inventioncan be fused to a constant region (e.g., hinge, CH2, and CH3 domains) ofhuman IgG1 or IgM molecule, (for example, as described by Hudson &Souriauso (2003) Nature Medicine 9(1):129-134) so as to make the fusedpolypeptides or fragments thereof more soluble and stable in vivo. Theshort half-life of antibody fragments can also be extended by‘pegylation’, that is, a fusion to polyethylene glycol (see Leong, S. R.et al. (2001) Cytokine 16:106-119). In one example of such fusions,described in WO0183525, Fc domains are fused with biologically activepeptides. A pharmacologically active compound is produced by covalentlylinking an Fc domain to at least one amino acid of a selected peptide.Linkage to the vehicle increases the half-life of the peptide, whichotherwise could be quickly degraded in vivo

Alternatively, non-classical alternative protein scaffolds (for examplesee Nygren & Skerra (2004) J Immunol Methods 290(1-2):3-28 orWO03049684) can be used to incorporate, and replicate the properties of,the peptides of the invention, for example by inserting peptidesequences derived from TREM-1 CDR2 or CDR3 into a protein framework tosupport conformationally variable loops having structural/functionalsimilarities to CDR2 or CDR3 in a fixed spatial arrangement

Such fusion proteins or scaffold based proteins can be used as animmunogen for the production of specific antibodies which recognize thepolypeptides of the invention or fragments thereof. In another preferredembodiment, such fusion proteins or scaffold based proteins can beadministered to a subject so as to inhibit interactions between a ligandand its receptors in vivo. Such inhibition of the interaction will blockor suppress certain cellular responses involved in sepsis and septicshock.

In one aspect, the fusion protein comprises a polypeptide of theinvention which is fused to a heterologous signal sequence at itsN-terminus. Various signal sequences are commercially available. Forexample, the secretory sequences of melittin and human placentalalkaline phosphatase (Stratagene; La Jolla, Calif.) are available aseukaryotic heterologous signal sequences. As examples of prokaryoticheterologous signal sequences, the phoA secretory signal (Sambrook, etal., supra; and Current Protocols in Molecular Biology, 1992, Ausubel,et al., eds., John Wiley & Sons) and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.) can be listed. Another example isthe gp67 secretory sequence of the baculovirus envelope protein (CurrentProtocols in Molecular Biology, 1992, Ausubel, et al., eds., John Wiley& Sons).

In another embodiment, a polypeptide of the invention can be fused totag sequences, e.g., a hexa-histidine peptide, such as the tag providedin a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311), among others, many of which are commercially available. Asdescribed in Gentz, et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824,for instance, hexa-histidine provides for convenient purification of thefusion protein. Other examples of peptide tags are the hemagglutinin“HA” tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, et al., 1984, Cell 37:767) and the “flag”tag (Knappik, et al., 1994, Biotechniques 17(4):754-761). These tags areespecially useful for purification of recombinantly producedpolypeptides of the invention.

Fusion proteins can be produced by standard recombinant DNA techniquesor by protein synthetic techniques, e.g., by use of a peptidesynthesizer. For example, a nucleic acid molecule encoding a fusionprotein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Current Protocols in Molecular Biology, 1992,Ausubel, et al., eds., John Wiley & Sons). The nucleotide sequencecoding for a fusion protein can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. Various host-vector systems and selection systems are known.In a specific embodiment, the expression of a fusion protein isregulated by a constitutive promoter. In another embodiment, theexpression of a fusion protein is regulated by an inducible promoter. Inaccordance with these embodiments, the promoter may be a tissue-specificpromoter. Expression vectors containing inserts of a gene encoding afusion protein can be identified by three general approaches: (a)nucleic acid hybridization, (b) presence or absence of “marker” genefunctions, and (c) expression of inserted sequences. In the firstapproach, the presence of a gene encoding a fusion protein in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to an inserted geneencoding the fusion protein. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain “marker” gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof a nucleotide sequence encoding a fusion protein in the vector. Forexample, if the nucleotide sequence encoding the fusion protein isinserted within the marker gene sequence of the vector, recombinantscontaining the gene encoding the fusion protein insert can be identifiedby the absence of the marker gene function. In the third approach,recombinant expression vectors can be identified by assaying the geneproduct (i.e., fusion protein) expressed by the recombinant. Such assayscan be based, for example, on the physical or functional properties ofthe fusion protein in in vitro assay systems, e.g., binding withanti-fusion protein antibody. For long-term, high-yield production ofrecombinant proteins, stable expression is preferred. For example, celllines which stably express the fusion protein may be engineered. Ratherthan using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedium, and then are switched to a selective medium. The selectablemarker in the recombinant plasmid confers resistance to the selectionand allows cells to stably integrate the plasmid into their chromosomesand grow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell linesthat express the differentially expressed or pathway gene protein. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of thedifferentially expressed or pathway gene protein. Once a fusion proteinof the invention has been produced by recombinant expression, it may bepurified by any method known in the art for purification of a protein,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antibody, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

The present invention also provides methods for treating a subjectsuffering from sepsis, septic shock or a sepsis-like condition byadministering a peptide or polypeptide of the invention. In anotherembodiment, the modulator may be an antibody which mimics the activityof a polypeptide of the invention. In particular, the invention providesa method of treating or ameliorating sepsis, septic shock or asepsis-like condition in a subject, comprising: administering atherapeutically effective amount of a peptide or polypeptide of any oneof the preceding claims to a subject. In such methods, the peptide orpolypeptide administered can have substantial sequence identity tosequence SEQ ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19, is SEQ ID NOS: 3, 4,6, 7, 16, 17, 18 or 19, or an active fragment, analogue or derivative ofSEQ ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19 or has at least about 80%sequence identity to SEQ ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19

In one aspect, the invention provides a method for preventing sepsis,septic shock or sepsis-like conditions, by administering to the subjecta peptide or polypeptide of the invention. Subjects at risk of sepsis orseptic shock can be identified by, for example, any diagnostic orprognostic assays as known in the art (for particularly suitable methodsof diagnosis, see WO2004081233, Gibot et al. (2004) Ann Intern Med.141(1):9-15 and Gibot et al. (2004) N Engl J Med. 350(5):451-8. Theprophylactic agents described herein, for example, can be used to treata subject at risk of developing disorders such as those previouslydiscussed. The methods of the invention are applicable to mammals, forexample humans, non human primates, sheep, pigs, cows, horses, goats,dogs, cats and rodents, such as mouse and rat. Generally, the methods ofthe invention are to be used with human subjects.

Furthermore, the invention provides a pharmaceutical compositioncomprising a polypeptide of the present invention or an antibody orfragments thereof that mimics a polypeptide of the invention. Thepeptides, polypeptides and antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the peptide, protein, or antibody and apharmaceutically acceptable carrier.

As used herein the language “pharmaceutically acceptable diluent,carrier or excipient” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The invention includes methods for preparing pharmaceutical compositionscontaining a peptide or polypeptide of the invention. Such compositionscan further include additional active agents. Thus, the inventionfurther includes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with a peptide orpolypeptide of the invention and one or more additional activecompounds.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, transdermal (topical), transmucosal, intra-articular,intraperitoneal, and intrapleural, as well as oral, inhalation, andrectal administration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy injectability with a syringe exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient, such as starch or lactose; a disintegratingagent, such as alginic acid, Primogel, or corn starch; a lubricant, suchas magnesium stearate or Sterotes; a glidant, such as colloidal silicondioxide; a sweetening agent, such as sucrose or saccharin; or aflavoring agent, such as peppermint, methyl salicylate, or orangeflavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described byCruikshank, et al., 1997, J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193).

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention further provides a kit containing a peptide or polypeptideof the invention of the present invention, or an antibody or fragmentsthereof mimicking a polypeptide of the invention, preferably withinstructions for use, for example in the treatment of sepsis, septicshock or sepsis-like conditions.

The invention provides a method for identifying (or screening)modulators, i.e., candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other drugs) which mimic apolypeptide of the invention or have a stimulatory or inhibitory effecton, for example, activity of a polypeptide of the invention. Inparticular, the invention provides a method of screening compounds orcompositions to treat sepsis, septic shock or sepsis-like conditions,comprising: providing a TREM-1 peptide; contacting an animal in a cecalligation and puncture model (or using other assay or model as describedherein or known in the art) with the TREM-1 peptide; determining ifthere was a modulation in the sepsis, for example wherein an increase insurvival indicates that the TREM-1 peptide may be useful for treatingsepsis, septic shock or sepsis-like conditions.

The invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

Preferred features of each aspect of the invention are applicable toeach other aspect, mutatis mutandis.

The present invention will now be described with reference to thefollowing non-limiting examples, with reference to the figures, inwhich:

FIG. 1A. shows a sequence alignment of TREM-1 and TREM-2 family members.Human TREM-1 (SEQ ID NO:1) was aligned with mouse TREM-1 (SEQ ID NO:2)and human and mouse TREM-2 (SEQ ID NO:26 and 27, respectively in orderof appearance) using version 1.74 of CLUSTAL W. Secondary structureassignments correspond to the published human TREM-1 structure (arrowsfor β-strands and cylinder for a helices) (Radaev et al. (2003)Structure (Camb). December; 11(12):1527-35). Residues involved inhomo-heterodimer formation are shown in white on black background.Cysteine making disulfide bonds conserved for V-type Ig fold are inbold. Gaps are indicated with (−), identical residues with (*), similarwith (: or .). An extended region of similarities between human andmouse TREM1 sequences is shown in boxes on grey background. TREM-1peptide sequences used in the Examples herein are indicated underlined.

FIG. 1B. shows a ribbon diagram of the published TREM-1 homodimericstructure (Kelker, et al. (2004) J Mol Biol. September 24;342(4):1237-48). Postulated binding sites that comprise the antibodyequivalent Complementarity Determining Regions (CDRs) are in red.

FIG. 2. shows that administration of TREM-1 peptides, 1 hour before LPS,reduces death induced by endotoxaemia. BALB/c mice (10 per group) wereinjected intraperitoneally with 200 μg LPS. The TREM-1 peptides P1, P2,P3, or P5 (200 μl of a 300 μM solution per mouse) were injectedintraperitoneally 1 hour before LPS. Viability of mice was monitoredtwice a day for 7 days. Statistical analysis was performed by Logranktest. Data from control mice represent cumulative survival curves fromtwo independent experiments performed under identical conditions.

FIG. 3. shows that TREM-1 peptide P1 is able to effectively reduce deathinduced by endotoxaemia when injected at 4 hours after LPS. BALB/c mice(10 per group) were injected intraperitoneally with 200 μg LPS. TREM-1peptide P1, 200 μl of a 300 μM solution per mouse was injectedintraperitoneally 1 hour before or 4 hours after LPS. Viability of micewas monitored twice a day for 7 days. Statistical analysis was performedwith the Logrank test. Data from control mice represent cumulativesurvival curves from two independent experiments performed underidentical conditions

FIG. 4. shows that administration of TREM-1 peptides, 4 hours after LPS,reduces death induced by endotoxaemia. BALB/c mice (10 per group) wereinjected intraperitoneally with 200 μmg LPS. P1 peptide, 200 μl of a150, 300 and 600 μM solution per mouse (dots) or P3, 200 μl of a 600 μMsolution per mouse (filled squares) were injected intraperitoneally 4hours after LPS. Viability of mice was monitored twice a day for 7 days.Statistical analysis was performed with the Logrank test

FIG. 5. shows that TREM-1 peptide P1 protects against cecal ligation andpuncture (CLP). CLP was induced in C57BL/6 mice (15 per group) asdescribed in Materials and Methods. P1 peptide (empty dots) or P3peptide (filled squares) (200 μl of a 600 μM solution per mouse) wereinjected intraperitoneally 5 and 24 hours after CLP induction. Viabilityof mice was monitored twice a day for 10 days. Statistical analysis wasperformed with the Logrank test.

FIG. 6. shows that P1, P2 and P5 peptides, but not P3 peptide, inhibitthe binding of soluble TREM-1/IgG1 to TREM-1 Ligand positive peritonealexudate cells. Cytofluorimetric analysis of peritoneal exudate cellswith 2 μg/ml of mouseTREM-1/hIgG1 in the presence of a 500 μM solutionper mouse (thin line), 100 μM solution per mouse (dotted line) orabsence (thick line) of the peptides is shown. The grey histogramrepresents immunostaining with human IgG1 as a control.

FIG. 7A. shows the release of sTREM-1 from cultured monocytes afterstimulation with LPS with and without proteases inhibitor. LPSstimulation induced the appearance of a 27-kD protein that wasspecifically recognized by an anti-TREM-1 mAb (inset). sTREM-1 levels inthe conditioned culture medium were measured by reflectance ofimmunodots. Data are shown as mean±SD (n=3).

FIG. 7B. shows expression of TREM-1 mRNA in monocytes. Culturedmonocytes were stimulated with LPS (1 μg/mL) for 0, 1 and 16 hours asindicated. LPS induced TREM-1 mRNA production within 1 hour.

FIG. 8A. shows the release of cytokines and sTREM-1 from culturedmonocytes. For cell activation, primary monocytes were cultured in24-well flat-bottom tissue culture plates in the presence of LPS (1μg/mL). In some experiments this stimulus was provided in combinationwith P5 (10 to 100 ng/mL), control peptide (10 to 100 ng/mL) or rIL-10(500 U/mL). To activate monocytes through TREM-1, an agonist anti-TREM-1mAb (10 μg/mL) was added as indicated. Cell-free supernatants wereanalysed for production of TNF-α, IL-1β and sTREM-1 by ELISA orimmunodot. All experiments were performed in triplicate and data areexpressed as means (SEM).

a: Media

b: P5 10 ng/mL

c: Anti-TREM-1

d: LPS

e: LPS+Anti-TREM-1

f: LPS+P5 10 ng/mL

g: LPS+P5 50 ng/mL

h: LPS+P5 100 ng/mL

i: LPS+IL10

FIG. 8B. shows the effect of P5 on NFκKB activation. Monocytes werecultured for 24 hours in the presence of E.coli LPS (O111:B4, 1 μg/mL),anti-TREM-1 mAb (10 μg/mL) and/or P5 (100 ng/mL) as indicated and thelevels of NFκB p50 and p65 were determined using an ELISA-based assay.Experiments were performed in triplicate and data are expressed as meansof optical densities (SEM).

FIG. 9. shows accumulation of sTREM-1 in serum of LPS-treated mice. MaleBalb/C mice (20 to 23 g) were treated with LPS (LD₅₀,intraperitoneally). Serum was assayed for sTREM-1 by immunodot. SerumsTREM-1 was readily detectable 1 hour after LPS administration and wasmaintained at a plateau level from 4 to 6 hours.

FIG. 10A. shows that P5 pre-treatment protects against LPS lethality inmice. Male Balb/C mice (20 to 23 g) were randomly grouped (10 mice pergroup) and treated with an LD₁₀₀ of LPS. P5 (50 μg or 100 μg) or controlvector was administered 60 min before LPS.

FIG. 10B. shows that delayed administration of P5 protects LPS lethalityin mice. Male Balb/C mice (20 to 23 g) were randomly grouped (8 mice pergroup) and treated with an LD₁₀₀ of LPS. P5 (75 μg) or control vectorwas administered 4 or 6 hours after LPS as indicated.

FIG. 10C. shows that administration of agonist TREM-1 mAb is lethal tomice. Male Balb/C mice (20 to 23 g) were randomly grouped (8 mice pergroup) and treated with a combination of an LD₅₀ of LPS+control vector,LD₅₀ of LPS+anti-TREM-1 mAb (5 μg) or LD₁₀₀ of LPS+control vector asindicated. Control vector and anti-TREM-1 mAb were administered 1 hourafter LPS injection

FIG. 11A. shows that P5 partially protects mice from CLP-inducedlethality. Male Balb/C mice (20 to 23 g) were randomly grouped andtreated with normal saline (n=14) or the control peptide (n=14, 100 μg)or with P5 (100 μg) in a single infection at H0 (n=18), H+4 (n=18) orH+24 (n=18). The last group of mice (n=18) was treated with repeatedinjections of P5 (100 μg) at H+4, H+8 and H+24.

FIG. 11B. shows the dose effect of P5 on survival. Mice (n=15 per group)were treated with a single injection of normal saline or 10 μg, 20 μg,50 μg 100 μg or 200 μg of P5 at H0 after the CLP and monitored forsurvival

FIG. 12. shows that P5 has no effect on bacterial counts during CLP.Mice (5 per group) were killed under anaesthesia at 24 hours after CLP.Bacterial counts in peritoneal lavage fluid and blood were determinedand results are expressed as CFU per mL of blood and CFU per mouse forthe peritoneal lavage.

FIG. 13 shows TNF-α and IL-1β plasma concentration evolution after LPS(15 mg/kg) administration in rats.

*p<0.05 P5-treated vs Control animals

^(§)p<0.05 P5-treated vs P1-treated animals

FIG. 14 shows Nitrite/Nitrate concentrations evolution after LPS (15mg/kg) administration in rats. *p<0.05 P5-treated vs Control andP1-treated animals

FIG. 15 shows mean arterial pressure evolution during caecal ligationand puncture-induced peritonitis in rats.

*p<0.05 vs Control animals

FIG. 16 shows TNF-α plasma concentration evolution during caecalligation and puncture-induced peritonitis in rats.

*p<0.05 P5-treated vs Control animals

^(§)p<0.05 P1-treated vs Control animals

^($)p<0.05 P5 vs P1-treated animals

FIG. 17 shows Nitrite/Nitrate concentration evolution during caecalligation and puncture-induced peritonitis in rats.

*p<0.05 P5 and P1-treated vs Control animals

FIG. 18 shows that hP5 efficiently protects mice from LPS induced septicshock (see Example 5). Septic shock was induced in male Balb/c mice(n=15/group) with 200 mg of LPS as described in Material and Methods.hP5 peptide or control were administered at the following time points:−1 h, 0 h, +1 h+4 h. Mortality was followed twice a day for 7 days. Datawere analysed using GraphPad Prism Survival curve analysis. Log-rank(Mantel-Cox) test showed a statistically significant difference betweenthe two curves (p=0.0003).

FIG. 19. shows the results for male C57BL/6 mice (n=15/group) that weresubject to Cecal Ligation and Puncture as described in Example 6 andtreated with hP5 peptide or scrambled peptide control at the followingtime points: −1 h, 0 h, +4 h and +24 h. Mortality was followed twice aday for 10 days. Data were analyzed using GraphPad Prism Survival curveanalysis.

FIG. 20. Disease Activity Index (DAI). Colitis was induced by oraladministration of DSS (see Example 7). Upon colitis induction, mice(n=8) were daily treated either with hP5 or its scrambled controlpeptide. Animal weight, haemoccult or presence of gross blood and stoolconsistency were used to determine the DAI score as indicated in Example7. *(grey)=DSS+hP5 versus DSS+scrambled peptide; *(black)=DSS+hP5 vsDSS+vehicle; ***p≦0.001, **p≦0.01, *p≦0.05.

FIG. 21. Body weight loss. Colitis was induced by oral administration ofDSS (see Example 7). Upon colitis induction, mice were daily treatedeither with hP5 or its scrambled control peptide. Weight was dailymeasured and percentage of weight loss versus day 0 was calculated.*(grey)=DSS+hP5 versus DSS+scrambled peptide; *(black)=DSS+hP5 vsDSS+vehicle; ***p≦0.001, **p≦0.01.

FIG. 22. Haemoccult or presence of gross blood in the feces. Colitis wasinduced by oral administration of DSS (see Example 7). Upon colitisinduction, mice were daily treated either with hP5 or its scrambledcontrol peptide. Haemoccult or presence of gross blood was detected andscore assigned as measured and score assigned at the indicated timepoints as described in. Example 7. *(grey)=DSS+hP5 versus DSS+scrambledpeptide; *(black)=DSS+hP5 vs DSS+vehicle; ***p≦0.001, **p≦0.01, *p≦0.05.

FIG. 23. Extent of diarrhea. Colitis was induced by oral administrationof DSS (see Example 7). Upon colitis induction, mice were daily treatedeither with hP5 or its scrambled control peptide. Stool consistency wasmeasured and score assigned at the indicated time points as described inExample 7. *(grey)=DSS+hP5 versus DSS+scrambled peptide;*(black)=DSS+hP5 vs DSS+vehicle; ***p≦0.001, **p≦0.01, *p≦0.05.

FIG. 24. Shortening of the colon. Colitis was induced by oraladministration of DSS (see Example 7). Upon colitis induction, mice weredaily treated either with hP5 or its scrambled control peptide. At day11 mice were euthanized and the colon length of each mouse measured fromthe anus to the end of the cecum. ***p≦0.001, **p≦0.01, *p≦0.05.

EXAMPLE 1 TREM-1 Peptides Protect Mice from Death by Septic Shock

TREM-1 peptides matching the following criteria were synthesized: i)highest homology between human and mouse TREM-1 and lowest homology withTREM-2. ii) peptides spanning the Complementarity Determining Regions(CDRs) of TREM-1. According to the published crystal structure ofTREM-1, and in analogy with antibodies, these residues are likely to beinvolved in cognate ligand recognition (Radaev et al. (2003) Structure(Camb). December; 11(12):1527-35 & Kelker, et al. (2004) J Mol Biol.September 24; 342(4):1237-48) (see FIG. 1). One peptide (P1) wasdesigned in the CDR2 region and three peptides (P2, P4 and P5) in theCDR3 region. A fourth peptide (P3) was designed in the neck regionconnecting the V-type immunoglobulin OM-like domain (Ig-V) to thetrans-membrane domain. No peptide was designed in the CDR1 region due tohigh sequence homology between TREM-1 and TREM-2.

Thus, the following peptides of the TREM-1 protein were ordered from andwere synthesized and purified by the Protein and Peptide ChemistryFacility, Institute of Biochemistry, University of Lausanne:

P1 (CDR2 67-89) LVVTQRPFTRPSEVHMGKFTLKH [SEQ ID NO: 3] P2 (CDR3 114-136)VIYHPPNDPVVLFHPVRLVVTKG [SEQ ID NO: 4] P3 (neck region 168-184)TTTRSLPKPTAVVSSPG [SEQ ID NO: 5] P4 (CDR3 103-123) LQVTDSGLYRCVIYHPPNDPV[SEQ ID NO: 6] P5 (CDR3 103-119): LQVTDSGLYRCVIYHPP [SEQ ID NO: 7]P1sc* (P1 scrambled seq.) LTPKHGQRSTHVTKFRVFEPVML [SEQ ID NO: 8]P5sc* (P5 scrambled seq.) TDSRCVIGLYHPPLQVY [SEQ ID NO: 9]*This is a control peptide and indeed does not protect

In the experiments of this example, the peptides were administered in avolume of 200 μl of the solution molarity indicated. To assess theability of TREM1-peptides to protect mice from LPS-induced endotoxaemia,the Inventors administered peptides P1, P2, P3 and P5 (300 μM) 1 hourbefore a lethal dose of lipopolysaccharide (LPS) (FIG. 2). Lethality wasmonitored over time and compared with animals that had received controlinjections of vehicle alone. P5 injection confers maximal protection,with 90% of the animals still alive 7 days after LPS injection, ascompared with 10% of control mice (p<0.001). 60% of the P1-treated miceand 50% of the P2 treated mice survived endotoxaemia as compared with10% of control mice (p<0.01 and p<0.05 respectively). Interestingly, allP3-treated mice died within 4 days after LPS injection. These resultsindicate that peptides containing sequences of the extracellular portionof TREM-1 corresponding to the putative ligand binding site (CDR2 andCDR3) can protect mice from lethal shock.

In order to investigate whether TREM-1 peptide treatment could bedelayed until after the administration of LPS, the Inventors injectedthe peptides at 4 hours after LPS injection. Only in the case of P1,this delayed treatment conferred significant protection against a lethaldose of LPS (FIG. 3). 80% of the mice injected with P1 4 hours after LPSsurvived endotoxaemia compared to 60% of mice treated 1 h before LPS and10% of mice treated with vehicle alone (p<0.001 and p<0.01respectively). Thus, P1 is effective even when injected after theoutbreak of endotoxaemia. No late death occurred over one week,indicating that P1 did not merely delay the onset of LPS lethality, butprovided lasting protection. P1 administration conferred maximalprotection (80%) when administered at 600 μM (p<0.01) and the level ofprotection dropped to 50% at 300 μM (p<0.05) and further down to 30% at150 μM as compared to 20% of control mice, indicating a dose dependenteffect of P1 (FIG. 4). The Inventors then investigated whether P1protects against septic shock in the “CLP” model (Cecal Ligation andPuncture is a widely used experimental model of sepsis). Mice treatedwith two doses of P1 at 5 and 24 hours after CLP were protected fromdeath as compared to control treated mice (p=0.0791) although thedifference was not statistically significant. 40% of the mice injectedwith P1 at 5 days after CLP survived compared to 5% of mice treated withP3 peptide. At 10 days after CLP, the treated mice were still alive,indicating that P1 did not merely delay mortality, but provided lastingprotection (FIG. 5).

EXAMPLE 2 TREM-1 Peptide P1 Inhibits Binding of Soluble Mouse TREM-1/IgGto TREM-1 Ligand Positive Cells

Among TREM-1 derived peptides tested in CLP, peptides P1, P2 and P5demonstrate a protective activity. A possible mechanism of action couldbe the ability of TREM-1 derived peptide to interfere with TREM-1/TREM-1ligand interaction. To address this question the Inventors performedcompetition experiments on TREM-1 ligand positive cells: PEC (PeritonealExudate Cells) from CLP treated mice.

Peritoneal exudate cells (PEC) from mice suffering from a caecalligation and puncture (CLP)-induced peritonitis were subjected to flowcytometry analysis after incubation with a PE-conjugated anti-human IgG1(Jackson Immunoresearch, Bar Harbor, USA). Competition with TREM-1peptides was performed by pre-incubating cells with the indicatedconcentrations of peptides for 45 minutes on ice before addingmTREM-1-IgG1.

As shown in FIG. 6, the P1 peptide, derived from the CDR2 region ofmTREM-1, and the P2 and P5 peptides spanning the CDR3 region inhibitTREM-1 interaction with its ligand in a dose dependent manner.Conversely the P3 peptide, derived from the neck region of TREM-1connecting the IgG like portion to the transmembrane domain wasineffective.

EXAMPLE 3 Additional Studies on the Modulation of the InflammatoryResponse in Murine Sepsis by TREM-1 Peptide P5

Methods

Preparation of Monocytes from Peripheral Blood

Ten mL of peripheral blood samples were collected on EDTA-K from 5healthy volunteer donors originating from laboratory staff. Afterdilution in RPMI (Life Technologies, Grand Island, N.Y.) v/v, blood wascentrifuged for 30 min at room temperature over a Ficoll gradient(Amersham Pharmacia, Uppsala, Sweden) to isolate PBMC. The cellsrecovered above the gradient were washed and counted. In order todeplete the suspensions of lymphocytes, cells were then plated in24-well flat-bottom tissue culture plates (Corning, Corning, N.Y.) at aconcentration of 5×10⁶/mL and allowed to adhere during 2 hours at 37° C.The resulting lymphocyte suspension was discarded and the adheringmonocytic cells were maintained in a 5% CO2 incubator at 37° C. incomplete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM Penicillin, 50μg/mL Streptomycin; Life Technologies) supplemented with 10% FCS(Invitrogen, Cergy, France).

TREM-1 Peptide

Using the human TREM-1 sequence in Gen-Bank, accession #AF287008 and themouse TREM-1 sequence #AF241219, a peptide “P5” (LQVTDSGLYRCVIYHPP; [SEQID NO:7]; was chemically synthesized as a C-terminally amidated peptide(Pepscan Systems, Lelystad, The Netherlands). The correct peptide wasobtained in greater than 99% yield and with measured mass of 1961 Daversus a calculated mass of 1962 Da and was homogeneous afterpreparative purification, as confirmed by mass spectrometry and analyticreversed phase-high performance liquid chromatography. A peptide “P5sc”containing the same amino-acids than P5 but in a different sequenceorder (TDSRCVIGLYHPPLQVY; [SEQ ID NO:9]) was similarly synthesized andserved as ‘control peptide’.

In vitro Stimulation of Monocytes

For activation, monocytes were cultured in the presence of E.coli LPS(O111:B4, 1 μg/mL, Sigma-Aldrich, La Verpillière, France). Cellviability was assessed by trypan blue exclusion and by measuring lactatedehydrogenase release. In some experiments, this stimulus was given incombination with TNF-α (5 to 100 ng/mL, R&D Systems, Lille, France),

IL-1β (5 to 100 ng/mL, R&D Systems), rIFN-γ (up to 100 U/mL, R&DSystems), rIL-10 (500 U/ml, R&D Systems) or up to 100 ng/mL of P5 orcontrol peptide. In order to activate monocytes through TREM-1, ananti-TREM-1 agonist monoclonal antibody (R&D Systems) was added asfollows: flat-bottom plates were precoated with 10 μg/mL anti-TREM-1 perwell. After thorough washing in phosphate buffered saline (PBS), themonocyte suspensions were added at a similar concentration as above.Some experiments were performed in the presence of protease inhibitors(PMSF and Protease Cocktail Inhibitor; Invitrogen). Cell-freesupernatants were assayed for the production of TNF-α and IL-1β by ELISAaccording to the recommendations of the manufacturer (BD Biosciences,San Diego, USA). To address the effect of P5 on NF-κB activity inmonocytes, an ELISA-based assay was performed (BD Mercury™ TransfactorKit, BD Biosciences). Monocytes were cultured for 24 hours in thepresence of E.coli LPS (O111:B4, 1 μg/mL), and/or an agonist anti-TREM-1monoclonal antibody (10 μg/mL), and/or P5 (100 ng/mL). Whole-cellextracts were then prepared and levels of NF-κB p50 and p65 weredetermined according to the recommendations of the manufacturer. Allexperiments were performed in triplicate and data are expressed as means(SEM).

Identification and Quantitation of sTREM-1 Release

Primary monocytes suspensions were cultured as described above. Thecells were treated with E.coli LPS (O111:B4, 1 μg/mL) for 24 hours at37° C. Cell-conditioned medium was submitted to Western-blotting usingan anti-TREM-1 monoclonal antibody (R&D Systems) in order to confirm thepresence of 27 kDa material recognized by anti-TREM-1. Soluble TREM-1levels were measured by assessing the optical intensity of bands onimmunodots by means of a reflectance scanner and the Quantity OneQuantitation Software (Bio-Rad, Cergy, France) as reported elsewhere(18). Soluble TREM-1 concentration from each sample was determined bycomparing the optical densities of the samples with reference tostandard curves generated with purified TREM-1. All measurements wereperformed in triplicate. The sensitivity of this technique allows thedetection of sTREM-1 levels as low as 5 pg/mL.

TREM-1 RT-PCR

Total mRNA was extracted from primary monocytes cultured in the presenceof LPS using a TRIzol reagent (Invitrogen), and reverse transcribedusing Superscript RT II (Invitrogen) to generate cDNA. RT-PCR conditionsthen used for all reactions were 94° C., 30 s/65° C., 30 s/68° C., 1 minfor 30 cycles. Amplification was performed with 2.5 mM MgCl2, 0.2 mMdNTP, 2.0 U Taq polymerase, and 20 pM 5′ and 3′ oligonucleotide primers(Proligos, Paris, France).

The sequences of the 5′ and 3′ primer pairs used were the following:

for TREM-1 (17) TTGTCTCAGAACTCCGAGCTGC;  [SEQ ID NO: 10] andGAGACATCGGCAGTTGACTTGG; [SEQ ID NO: 11] for TREM-1sv (19)GGACGGAGAGATGCCCAAGACC;  [SEQ ID NO: 12] and ACCAGCCAGGAGAATGACAATG; [SEQ ID NO: 13] for β-actin (used as housekeeping amplicon)GGACGACATGGAGAAGATCTGG;  [SEQ ID NO: 14] and ATAGTAATGTCACGCACGATTTCC; [SEQ ID NO: 15]PCR products were run on agarose gels and visualized by ethidium bromidestaining.LPS-Induced Endotoxinemia in Mice

After approval by the local ethical committee, male Balb/C mice (20 to23 g) were randomly grouped and treated with E.coli LPSintraperitoneally (i.p.) in combination with P5 (in 500 μl normalsaline) or control vector before or after LPS challenge. In someexperiments, 5 μg of an anti TREM-1 monoclonal antibody was administeredi.p. one hour after LPS injection. The viability of mice was examinedevery hour, or animals were sacrificed at regular intervals. Serumsamples were collected by cardiac puncture and assayed for TNF-α andIL-1β by ELISA (BD Biosciences), and for sTREM-1 levels by immunodot.

CLP Polymicrobial Sepsis Model

Male Balb/C mice (7 to 9 weeks, 20 to 23 g) were anaesthetized by i.p.administration of ketamine and xylazine in 0.2 mL sterile pyrogen-freesaline. The caecum was exposed through a 1.0 cm abdominal midlineincision and subjected to a ligation of the distal half followed by twopunctures with a G21 needle. A small amount of stool was expelled fromthe punctures to ensure patency. The caecum was replaced into theperitoneal cavity and the abdominal incision closed in two layers. Aftersurgery all mice were injected s.c. with 0.5 ml of physiologic salinesolution for fluid resuscitation and s.c. every 12 h with 1.25 mg (i.e.50 μg/g) of imipenem. The animals were randomly grouped and treated withnormal saline (n=14), the control peptide (n=14, 100 μg) or P5 (100 μg)in a single injection at H0 (n=18), H+4 (n=18) or H+24 (n=18). The lastgroup of mice (n=18) was treated with repeated injections of P5 (100 μg)at H+4, H+8 and H+24. All treatments were diluted into 500 μl of normalsaline and administered i.p. The Inventors next sought to determine theeffect of various doses of P5. For this purpose, mice (n=15 per group)were treated with a single injection of normal saline or 10 μg, 20 μg,50 μg 100 μg or 200 μg of P5 at H0 after the CLP and monitored forsurvival. Five additional animals per group were killed underanaesthesia 24 hours after CLP for the determination of bacterial countand cytokines levels. Peritoneal lavage fluid was obtained using 2 mLRPMI 1640 (Life Technologies) and blood was collected by cardiacpuncture. Concentrations of TNF-α and IL-1β in the serum were determinedby ELISA (BD Biosciences). For the assessment of bacterial counts, bloodand peritoneal lavage fluid were plated in serial log dilutions ontryptic soy supplemented with 5% sheep blood agar plates. After plating,tryptic soy agar plates were incubated at 37° C. aerobically for 24hours, and anaerobically for 48 hours. Results are expressed as CFU permL of blood and CFU per mouse for the peritoneal lavage.

Statistical Analyses

Serum sTREM-1 and cytokines levels were expressed as mean (±SD). Theprotection against LPS lethality by P5 was assessed by comparison ofsurvival curves using the Log-Rank test. All statistical analyses werecompleted with Statview software (Abacus Concepts, Berkeley Calif.) anda two-tailed P<0.05 was considered significant.

Results

A Soluble Form of TREM-1 is Released from Cultured Human Monocytes afterStimulation with E.coli LPS

To identify the potential release of sTREM-1 in vitro, the Inventorsstimulated human monocytes with LPS and analyzed the conditioned culturemedium by SDS-PAGE. LPS stimulation induced the appearance of a 27-kDaprotein in a time-dependent manner (FIG. 7A). Western blotting analysisrevealed that this protein was specifically recognized by a monoclonalantibody directed against the extra-cellular domain of TREM-1 (FIG. 7A).Cell viability was unaffected at LPS concentrations that induced thepresence of sTREM-1 in conditioned medium, indicating that TREM-1release was not due to cell death. Similarly, treatment of monocyteswith protease inhibitors did not affect TREM-1 release (FIG. 7A). TREM-1mRNA levels were increased upon LPS treatment (FIG. 7B) whereas TREM-1svmRNA levels remained undetectable. This suggests that TREM-1 release islikely to be linked to an increased transcription of the gene andunrelated to TREM-1sv expression. Stimulation of monocytes for 16 hourswith TNF-α (5 to 100 ng/mL) or IL-1β (5 to 100 ng/mL) induced very smallTREM-1 release in a cytokine dose-dependent manner. Stimulation withIFN-γ did not induce TREM-1 release, even at concentrations of up to 100U/mL.

LPS Associated Release of Pro-Inflammatory Cytokines is Attenuated by P5

Significant TNF-α and IL-1β production was observed in the supernatantof monocytes cultured with LPS. TNF-α and IL-1β production was evenhigher for cells cultured with both TREM-1 mAb and LPS as compared withthose cultured with mAb or LPS alone (FIG. 8A).

The inducible release of pro-inflammatory cytokines was significantlylower after LPS stimulation when the medium was supplemented with P5 orIL-10. P5 reduced, in a concentration-dependent manner, the TNF-α andIL-1β production from cells cultured with LPS or with LPS and mAb andsimultaneously increased the release of sTREM-1 from cells cultured withLPS. The control peptide displayed no action on cytokines or sTREM-1release (data not shown). In striking contrast, IL-10 totally inhibitedthe release of both TREM-1 and inflammatory cytokines (FIG. 8A). BothLPS and TREM-1 mAb induced a strong activation of monocytic NF-κB p50and p65 and combined administration of LPS and TREM-1 mAb lead to asynergistic effect. P5 inhibited the NF-κB activation induced by theengagement of TREM-1 but did not alter the effect of LPS (FIG. 8B).

Serum sTREM-1 Levels of LPS-Treated Mice are Increased

In order to determine whether sTREM-1 was released systemically duringendotoxemia in mice, the Inventors measured serum sTREM-1 levels afterLPS administration. Serum sTREM-1 was readily detectable 1 hour afteradministration of an LD₅₀ dose of LPS and was maintained at peak plateaulevels from 4 to 6 hours after LPS treatment (FIG. 9).

TREM-1 Peptide “P5” Protects Endotoxemic Mice from Lethality

Mice treated by a single dose of P5 60 min before a lethal dose (LD₁₀₀)of LPS were prevented from death in a dose-dependent manner (FIG. 10A).In order to investigate whether P5 treatment could be delayed untilafter the administration of LPS, the Inventors injected P5 beginning 4or 6 hours after LPS injection. This delayed treatment up to 4 hoursconferred significant protection against a LD₁₀₀ dose of LPS (FIG. 10B).No late death occurred over one week, indicating that P5 did not merelydelay the onset of LPS lethality, but provided lasting protection.Control mice all developed lethargy, piloerection, and diarrhoea beforedeath. By contrast, P5-treated mice remained well groomed and active,had no diarrhoea, and were lively. To clarify the mechanism by which P5protected mice from LPS lethality, the Inventors determined the serumlevels of TNF-α, IL-1β and sTREM-1 of endotoxemic mice at 2 and 4 hours.As compared to controls, pre-treatment by 100 μg of P5 reduced cytokineslevels by 30% and increased sTREM-1 levels by 2 fold as shown in Table4:

TABLE 4 Serum concentrations of TNF-α, IL-1β and sTREM-1 in endotoxemicmice. TNF-α (ng/mL) IL-1β (ng/mL) sTREM-1 (ng/mL) H2 H4 H2 H4 H2 H4Control 3.3 ± 1.0 0.4 ± 0.1 0.3 ± 0.1 1.5 ± 0.2 249 ± 48 139 ± 8 P5 (100μg) 2.4 ± 0.5 0.1 ± 0.1 0.2 ± 0.1 0.9 ± 0.2 475 ± 37 243 ± 28

Engagement of TREM-1 is Lethal to Mice

To further highlight the role of TREM-1 engagement in LPS-mediatedmortality, mice were treated with agonist anti-TREM-1 mAb in combinationwith the administration of an LD₅₀ dose of LPS. This induced asignificant increase in mortality rate from 50% to 100% (FIG. 10C).

P5 Protects Mice from CLP-Induced Lethality

To investigate the role of P5 in a more relevant model of septic shock,the Inventors performed CLP experiments (FIG. 11A). The control groupscomprised mice injected with normal saline or with the control peptide.In this model of polymicrobial sepsis, P5 still conferred a significantprotection against lethality even when administered as late as 24 hoursafter the onset of sepsis. Interestingly, repeated injections of P5 hadthe more favourable effect on survival (P<0.01). There was a doseresponse effect of P5 on survival (FIG. 11B) and cytokine production(Table 5). P5 had no effect on bacterial clearance (FIG. 12).

TABLE 5 Serum concentrations of TNF-α, IL-1β and sTREM-1 at 24 hoursafter CLP. TNF-α (pg/mL) IL-1β (pg/mL) sTREM-1 (ng/mL) Control peptide105 ± 12  841 ± 204 52 ± 3 Control saline 118 ± 8   792 ± 198 35 ± 5 P510 μg 110 ± 11 356 ± 62 43 ± 8 P5 20 μg  89 ± 10 324 ± 58 58 ± 8 P5 50μg 24 ± 6  57 ± 11  93 ± 10 P5 100 μg 20 ± 3 31 ± 3 118 ± 12 P5 200 μg21 ± 7 37 ± 8 158 ± 13

Sepsis exemplifies a complex clinical syndrome that results from aharmful or damaging host response to severe infection. Sepsis developswhen the initial, appropriate host response to systemic infectionbecomes amplified, and then dysregulated (4, 5). Neutrophils andmonocyte/macrophages exposed to LPS, for instance, are activated andrelease such pro-inflammatory cytokines as TNF-α and IL-1β. Excessiveproduction of these cytokines is widely believed to contribute to themulti-organ failure that is seen in septic patients (20-23).

TREM-1 is a recently identified molecule involved in monocyticactivation and inflammatory response (12, 14). It belongs to a familyrelated to NK cell receptors that activate downstream signalling events.The expression of TREM-1 on PNNs and monocytes/macrophages has beenshown to be inducible by LPS (16, 17).

As described herein, the Inventors demonstrate that a soluble form ofTREM-1 was released from cultured human monocytes after stimulation withE.coli LPS. Such a soluble form was also detectable in the serum ofendotoxemic mice as early as 1 hour after LPS challenge. This isconsistent with the implication of TREM-1 in the very early phases ofthe innate immune response to infection (14, 15, 24). The mechanism bywhich sTREM-1 is released is not clearly elucidated but seems to berelated to an increased transcription of the TREM-1 gene. Nevertheless,although incubation with a protease inhibitor cocktail does not alterthe sTREM-1 release, cleavage of the surface TREM-1 from the membranecannot be totally excluded. Interestingly, stimulation of humanmonocytes with such pro-inflammatory cytokines as TNF-α, IL-1β or IFN-γinduced very small sTREM-1 release unless LPS was added as aco-stimulus. The expression of an alternative mRNA TREM-1 splice variant(TREM-1sv) has been detected in monocytes that might translate into asoluble receptor (18) upon stimulation with cell wall fraction ofMycobacterium bovis BCG but not LPS (25). This was confirmed in thisstudy as i) LPS did not increase the level of mRNA TREM-1sv in monocytesand ii) only a 27-kDa protein was released by monocytes upon LPSstimulation and not the 17.5-kDa variant.

Although its natural ligand has not been identified (13, 14), engagementof TREM-1 on monocytes with an agonist monoclonal antibody resulted in afurther enhancement of pro-inflammatory cytokines production, while P5induced a decrease of these syntheses in a concentration-dependentmanner, and IL-10 completely suppressed it.

Inflammatory cytokines, and especially TNF-α, are considered to bedeleterious, yet they also possess beneficial effects in sepsis (5) asshown by the fatal issue of peritonitis in animals with impaired TNF-αresponses (9-11). Moreover, in clinical trials, the inhibition of TNF-αincreased mortality (8). Finally, the role of TNF-α in the clearance ofinfection has been highlighted by the finding that sepsis is a frequentcomplication in rheumatoid arthritis patients treated with TNF-αantagonists (26).

The mechanism by which P5 modulates cytokine production is not yetclear. P5 comprises the complementary determining region (CDR)-3 and the‘F’ β strand of the extra-cellular domain of TREM-1. The latter containsa tyrosine residue mediating dimerization. Radaev et al postulated thatTREM-1 captures its ligand with its CDR-equivalent loop regions (27). P5could thus impair TREM-1 dimerization and/or compete with the naturalligand of TREM-1. Moreover, the increase of sTREM-1 release frommonocytes mediated by P5 could prevent the engagement of membraneTREM-1, sTREM-1 acting as a decoy receptor, as in the TNF-α system (28,29).

Activation of the transcription factor NF-κB is a critical step inmonocyte inflammatory cytokine production after exposure to bacterialstimuli such as LPS (30, 31). Among the various NF-κB/Rel dimers, thep65/p50 heterodimer is the prototypical form of LPS-inducible NF-κB inmonocytes (32). P5 abolishes the p65/p50 NF-κB over-activation inducedby the engagement of TREM-1. This might at least partially explain theeffects of P5 on cytokine production and the protection from lethalityshown here to occur when the peptide was injected one hour beforeLPS-induced septic shock, or even up to 4 hours after.

Endotoxemia is simple to achieve experimentally, but imperfectly suitedto reproduce human sepsis, while polymicrobial sepsis induced by CLP isa more complex but better model, including the use of fluidresuscitation and antibiotics. The latter was thus also used in thisstudy, and confirmed the dose-dependent protection provided by P5, evenwhen administered as late as 24 hours after the onset of sepsis. Thefavourable effect of P5 was however unrelated to an enhanced bacterialclearance.

One difficulty in the use of immunomodulatory therapies is that it isnot possible to predict the development of sepsis, and, thus, patientsreceiving those treatments frequently already have well-establishedsepsis (6). Since P5 appeared to be effective even when injected afterthe outbreak of sepsis, it could thus constitute a realistic treatment(24, 33).

By contrast, engagement of TREM-1 by an agonist anti-TREM-1 monoclonalantibody mediated a dramatic increase of mortality rate inLPS-challenged mice: this further underscores the detrimental effect ofTREM-1 engagement during septic shock.

Experimental septic shock reproduces human sepsis only in part. Indeed,our group recently showed that significant levels of sTREM-1 werereleased in the serum of critically ill patients with sepsis patients(34), the highest levels being observed in patients who survived. Thisis consistent with our experimental findings indicating that the moreimportant sTREM-1 release, the more favourable is the outcome, and thussustains, at least theoretically, the potential value of soluble TREMpeptides as post-onset sepsis therapy.

TREM-1 appears to be a crucial player in the immediate immune responsetriggered by infection. In the early phase of infection, neutrophils andmonocytes initiate the inflammatory response owing to the engagement ofpattern recognition receptors by microbial products (3, 4). At the sametime, bacterial products induce the up-regulation and the release ofsTREM-1. Upon recognition of an unknown ligand, TREM-1 activatessignalling pathways which amplify these inflammatory responses, notablyin monocytes/macrophages. The modulation of TREM-1 signalling reduces,although without complete inhibition, cytokine production and protectsseptic animals from hyper-responsiveness and death. Modulation of TREM-1engagement with such a peptide as P5 might be a suitable therapeutictool for the treatment of sepsis, particularly because it seems to beactive even after the onset of sepsis following infectious aggression.

EXAMPLE 4 Haemodynamic Studies in LPS Treated and Septic Rats Treatedwith P1 and P5

The role of TREM-1 peptides in further models of septic shock, wasinvestigated by performing LPS and CLP (caecal ligation and puncture)experiments in rats.

Materials and Methods

LPS-Induced Endotoxinemia

Animals were randomly grouped (n=10-20) and treated with Escherichiacoli LPS (O111:B4, Sigma-Aldrich, Lyon, France) i.p. in combination withthe TREM-1 or scrambled peptides.

CLP Polymicrobial Sepsis Model

The procedure has been described in details elsewhere (see Mansart, A.et al. Shock 19:38-44 (2003)). Briefly, rats (n=6-10 per group) wereanesthetized by i.p. administration of ketamine (150 mg/kg). The caecumwas exposed through a 3.0-cm abdominal midline incision and subjected toa ligation of the distal half followed by two punctures with a G21needle. A small amount of stool was expelled from the punctures toensure potency. The caecum was replaced into the peritoneal cavity andthe abdominal incision closed in two layers. After surgery, all ratswere injected s.c. with 50 mL/kg of normal saline solution for fluidresuscitation. TREM-1 or scrambled peptides were then administered asabove.

Haemodynamic Measurements in Rats

Immediately after LPS administration as well as 16 hours after CLP,arterial BP (systolic, diastolic, and mean), heart rate, abdominalaortic blood flow, and mesenteric blood flow were recorded using aprocedure described elsewhere (see Mansart, A. et al. Shock 19:38-44(2003)). Briefly, the left carotid artery and the left jugular vein werecannulated with PE-50 tubing. Arterial BP was continuously monitored bya pressure transducer and an amplifier-recorder system (IOX EMKATechnologies, Paris, France). Perivascular probes (Transonic Systems,Ithaca, N.Y.) wrapped up the upper abdominal aorta and mesentericartery, allowed to monitor their respective flows by means of aflowmeter (Transonic Systems). After the last measurement (4^(th) hourduring LPS experiments and 24^(th) hour after CLP), animals weresacrificed by an overdose of sodium thiopental i.v.

Biological Measurements

Blood was sequentially withdrawn from the left carotid artery. Arteriallactate concentrations and blood gases analyses were performed on anautomatic blood gas analyser (ABL 735, Radiometer, Copenhagen, Denmark).Concentrations of TNF-α and IL-1β in the plasma were determined by anELISA test (Biosource, Nivelles, Belgium) according to therecommendations of the manufacturer. Plasmatic concentrations ofnitrates/nitrites were measured using the Griess reaction (R&D Systems,Abingdon, UK).

Statistical Analyses

Results are expressed as mean±SD. Between-group comparisons wereperformed using Student't tests. All statistical analyses were completedwith Statview software (Abacus Concepts, Calif.) and a two-tailed P<0.05was considered significant.

Results

Endotoxinemia Model

Following LPS administration, arterial pressures, aortic and mesentericblood flows dropped rapidly in control animals (scrambled peptidestreated rats) while the heart rate remained unchanged (Table 6). Thedecrease of arterial pressures and aortic blood flow was delayed untilthe second hour in TREM-1 peptide treated animals with significantlyhigher values by that time than in control animals. There was nodifference between P1 and P5 treated groups. By contrast, none of thesetwo peptides had any effect on the decrease of the mesenteric blood flow(Table 6).

Arterial pH remained constant over time until the fourth hour after LPSinjection where it severely dropped in the control group only (Table 6).The significant arterial lactate level elevation present in controlanimals after the third hour was abolished by the TREM-1 peptides (Table6). There was no difference between P1 and P5 with regard to pH,arterial bicarbonate and lactate concentrations.

As expected, a peak of TNF-α plasmatic concentration was induced by LPSbetween 30 minutes and 1 hour after injection followed by a progressivedecline thereafter (FIG. 13A). P1 peptide injection had no effect onthis production, while P5 attenuated TNF-α production by ˜30%.

P1 delayed the IL-1β peak until the third hour after LPS injection, butwithout attenuation. By contrast, P5 strongly reduced IL-1β release(FIG. 13B).

Nitrite/nitrate concentrations increased rapidly after LPSadministration in control and P1 treated animals but remained stableupon P5 treatment (FIG. 14).

TABLE 6 Hemodynamic parameters during LPS-induced endotoxinemiaMesenteric Heart Rate MAP Aortic blood blood flow Lactate (bpm) (mmHg)flow (mL/min) (mL/min) pH (mmol/L) Control H0 486 ± 13 123 ± 21 45 ± 713.6 ± 3.4  7.31 ± 0.03  3.3 ± 0.8 H1 522 ± 16 103 ± 25 25 ± 8^(a) 9.6 ±3.3 7.28 ± 0.03  4.2 ± 0.3 H2 516 ± 13  98 ± 23 12 ± 5^(a,b) 8.0 ± 3.77.29 ± 0.03  5.9 ± 0.6 H3 490 ± 20  78 ± 8^(a,b)  8 ± 3^(a,b) 5.8 ± 1.17.26 ± 0.01  7.9 ± 1.8^(a,b) H4 510 ± 18  67 ± 9^(a,b)  6 ± 1^(a,b) 4.1± 0.8 7.03 ± 0.10^(a,b) 11.5 ± 0.7^(a,b) P1 H0 464 ± 25 116 ± 10 49 ± 1112.0 ± 3.7  7.32 ± 0.04  2.7 ± 0.1 H1 492 ± 26 119 ± 14 39 ± 12^(a) 10.5± 1.7  7.29 ± 0.04  4.9 ± 1.1 H2 492 ± 26 113 ± 21 26 ± 14^(a) 7.7 ± 2.77.30 ± 0.01  5.0 ± 0.9 H3 480 ± 30  97 ± 29^(a) 22 ± 8^(a) 5.0 ± 1.07.26 ± 0.06  5.7 ± 0.7^(a) H4 480 ± 20  92 ± 7^(a) 16 ± 6^(a) 4.8 ± 0.97.26 ± 0.08^(a)  7.9 ± 1.7^(a) P5 H0 474 ± 49 115 ± 16 48 ± 9 12.8 ±6.4  7.33 ± 0.04  3.4 ± 1.5 H1 498 ± 26  99 ± 22 32 ± 8 11.4 ± 2.7  7.28± 0.06  5.4 ± 1.4 H2 510 ± 42 101 ± 18 23 ± 4^(b) 9.2 ± 1.9 7.32 ± 0.07 5.5 ± 1.6 H3 517 ± 62  93 ± 21^(b) 20 ± 7^(b) 6.0 ± 0.8 7.29 ± 0.11 5.9 ± 1.7^(b) H4 510 ± 26  89 ± 10^(b) 15 ± 6^(b) 5.0 ± 1.0 7.28 ±0.12^(b)  7.4 ± 1.8^(b) ^(a)p < 0.05 P1 vs Controls ^(b)p < 0.05 P5 vsControlsCLP Model

As the severity of the Inventors' model was at its highest 16 to 20hours after the completion of the CLP, the Inventors chose toinvestigate animals by the 16^(th) hour. Importantly, there were nodeaths before this time point. Although all animals were fluidresuscitated, none received antibiotics in order to strictly considerthe role of the peptides.

There was a dramatic decline in arterial pressure in the control animalsover time, and by H24 systolic, diastolic and mean arterial pressureswere 58±7 mmHg, 25±4 mmHg and 38±2 mmHg respectively. This decrease wasalmost totally abolished with P1 or P5 treatment with no significantdifference between H16 and H24 (FIG. 15). There was no differencebetween P1 and P5 treated rats.

TREM-1 peptides also prevented the aortic and mesenteric blood flowsdecrease observed in control animals (Table 7). The protective effect onmesenteric blood flow alterations was even higher under P5 treatment.The relative preservation of blood flows was not related to an increasedheart rate, since the latter was rather slower than in control animals(Table 7).

The progressive metabolic acidosis that developed in control rats wasattenuated by the P1 peptide, and almost abrogated by P5. The sameprotective trend was observed for arterial lactate elevation with a morepronounced effect of P5 (Table 7).

TABLE 7 Hemodynamic and selected biochemical parameters during CLPpolymicrobial sepsis Aortic blood Mesenteric Heart Rate flow blood flowBicarbonate Lactate (bpm) (mL/min) (mL/min) pH (mmol/L) (mmol/L) ControlH16 516 ± 44^(a,b) 38 ± 10 10.6 ± 3.0^(b) 7.31 ± 0.07^(b) 16.9 ± 2.7 4.7 ± 1.5^(b) H20 543 ± 35^(a,b) 19 ± 11^(a,b)  4.3 ± 1.5^(b) 7.23 ±0.05^(a,b) 12.0 ± 5.6^(a,b)  8.5 ± 1.4^(a,b) H24 480 ± 20 14 ± 9^(a,b) 2.5 ± 0.7^(b) 7.17 ± 0.01^(a,b) 10.3 ± 3.3^(a) 10.8 ± 1.9^(a,b) P1 H16462 ± 16^(a) 41 ± 12 13.5 ± 7.2 7.32 ± 0.04 16.8 ± 4.4  4.9 ± 0.4 H20480 ± 30^(a) 28 ± 17^(a)  5.3 ± 3.0^(c) 7.31 ± 0.18^(a) 16.0 ± 5.4^(b) 5.3 ± 1.1^(a,c) H24 420 ± 30 22 ± 16^(a)  4.5 ± 2.1^(c) 7.24 ±0.06^(a,c) 11.2 ± 0.8^(c)  6.8 ± 0.9^(a,c) P5 H16 460 ± 17^(b) 41 ± 1415.3 ± 3.5^(b) 7.35 ± 0.01^(b) 18.6 ± 2.0  3.3 ± 0.4^(b) H20 500 ±17^(b) 31 ± 5^(b) 11.0 ± 6.9^(b,c) 7.34 ± 0.01^(b) 18.0 ± 0.9^(a)  3.6 ±0.9^(b,c) H24 510 ± 20 28 ± 8^(b)  8.5 ± 3.5^(b,c) 7.36 ± 0.01^(b,c)17.1 ± 0.9^(a,c)  4.9 ± 1.1^(b,c) ^(a)p < 0.05 P1 vs Controls ^(b)p <0.05 P5 vs Controls ^(c)p < 0.05 P5 vs P1

Both P1 and P5 induced a decrease in TNF-α production, again with astronger effect of P5. By H20, plasmatic TNF-α was almost undetectableunder P5 treatment whereas it remained elevated in the other groups ofanimals (FIG. 16).

Nitrite/nitrate concentrations were increased in control animals butremained at a low level in both TREM-1 peptides treated groups (FIG.17).

A protective action of both P5 and P1 on hemodynamics was thus observedin septic rats. Both arterial pressure and blood flows were preserved,independently of heart rate. Moreover, modulation of TREM-1 signallingreduced, although not completely, cytokine production and protectedseptic animals from hyper-responsiveness. The fact that the cytokineproduction was not totally inhibited is a crucial point. Indeed,although inflammatory cytokines such as TNF-α are considereddeleterious, they also display beneficial effects in sepsis asunderlined by the fatal issue of peritonitis models in animals withimpaired TNF-α responses.

The activation of iNOS observed during septic shock leads to theproduction of large amount of NO that partly explains some of theperipheral vascular disorders (notably vasodilation and hypotension). Onthe myocardium itself, most of the action of NO is mediated by anactivation of the soluble guanylate-cyclase responsible for theproduction of cGMP which impairs the effect of cytosolic calcium oncontraction. Cyclic GMP is also able to stimulate the activity of somephosphodiesterases. The subsequent decrease of intra-cellular cAMPlevels could explain the ability of NO to attenuate the effects of betaadrenergic stimulation. The preservation of arterial pressure couldtherefore be partly explained by a lessened production of NO, asreflected by the lower concentrations of plasma nitrite/nitrate inTREM-1 peptides treated animals.

The decrease in inflammatory cytokine production could partly explainthe effect noted on blood flows. Indeed, although the list of potentialcytokine mediators of myocardial depression is long, TNF-α and IL-1βhave been shown to be good candidates Both these latter cytokinesdepressed myocardial contractility in vitro or ex vivo. Moreover, theneutralization or removal of TNF-α or IL-1β from human septic serumpartly abrogates the myocardial depressant effect in vitro and in vivo.Although P1 and P5 had an identical action on blood flows and arterialpressure during endotoxinemia, their action on cytokine productiondiffered with only a slight effect of P1 on plasma TNF-α and IL-1βconcentrations. The protective role of the TREM-1 peptides couldtherefore be only partly related to their action on cytokine release, orinvolve redundant pathways.

The modulation of the TREM-1 pathway by the use of small syntheticpeptides had beneficial effects on haemodynamic parameters duringexperimental septic shock in rats, along with an attenuation ofinflammatory cytokine production.

In summary, these data show that the TREM-1 peptides of the invention 1)efficiently protect subject animals from sepsis-related hemodynamicdeterioration; 2) attenuate the development of lactic acidosis; 3)modulate the production of such pro-inflammatory cytokines as TNF-α andIL-1β and 4) decrease the generation of nitric oxide. Thus TREM-1peptides are potentially useful in the restoration of haemodynamicparameters in patients with sepsis, septic shock or sepsis-likeconditions and therefore constitute a potential treatment for theaforesaid conditions.

EXAMPLE 5 hP5 Activity in a Murine Model of Sepsis: Endotoxin-InducedSeptic Shock

The activity of human P5 (hP5) was investigated in a murine model. hP5differs from mP5 according to ClustalW comparison as set our below:

SeqA Name  Length (aa) SeqB Name Length (aa) Score 1 hP5 17 2 mP5 17 82hP5 LQVEDSGLYQCVIYQPP 17 mP5 LQVTDSGLYRCVIYHPP 17 *** *****:****:**

For the experiment, male BALB/c mice (19-21 g) were randomly grouped (15mice per group) and injected intraperitoneally (i.p.) with 200 microg ofLPS from E. coli 0111:B4 (Sigma). A blinded investigator performed allinjections. 200 microliters of TREM-1 peptides dissolved in water 10%DMSO, 9% Solutol were administered intraperitoneally at −1 h, 0 h, +1 h,+4 h prior and after LPS injection. Viability of treated mice wasmonitored twice a day for 7 days.

To then assess the ability of hP5 peptide to protect mice fromLPS-induced endotoxaemia, the inventors administered at −1 h, 0 h, +1 h,+4 h prior and after LPS injection a lethal dose of lipopolysaccharide(LPS) (FIG. 18). Lethality was monitored over time and compared withanimals that had received vehicle alone. hP5 injection confers highprotection, with 80% of the animals still alive 7 days after LPSinjection, as compared with no survivors in the control group(p<0.0003). The human P5 peptide shows >80% identity with mP5.

The results summarised in FIG. 18 clearly demonstrate that the human P5protects mice from lethal shock.

EXAMPLE 6 hP5 Activity in a Murine Model of Sepsis: Cecal Ligation andPuncture Model

For the experiment the mice underwent a standardized preparation forlaparotomy (anaesthesia with 2% inhaled isoflurane in oxygen, shavingwith animal clippers, alcohol scrub). A 1 cm incision was made on themidline. The cecum was exposed and will be tightly ligated at 50-80%over its base with a 4-0 silk suture avoiding bowel obstruction. Thececum was then punctured once with a 23G needle. The cecum was gentlysqueezed until feces be just visible through the puncture, and placedagain in the abdominal cavity. The incision was thereafter be closedwith a 4-0 silk. 200 μl of hP5 or its scrambled peptide control werefreshly dissolved in water 10% DMSO, 9% solutol and injectedintraperitoneally with a 22 G needle at the following time points: −1,0, +4, +24. No fluid resuscitation was administered. Survival andMoribundity were observed twice a day for 10 days.

The Inventors then analysed whether hP5 protects against septic shock inthe “CLP” model. Mice treated with four doses of hP5 at −1 h, 0 h, +4 hand +24 hours after CLP were protected from death as compared to controltreated mice. At 72 hours after CLP, 73.3% of the mice injected with hP5survived compared to 60% of mice treated with scrambled peptide. At 10days after CLP, 66.6% of the hP5 treated mice were still alive comparedto 60% of the control group, suggesting that hP5 could provide lastingprotection (FIG. 19).

EXAMPLE 7 The human TREM-1 Peptide P5 (hP5) Attenuates EstablishedIntestinal Inflammation in the DSS Colitis Model

The purpose of this example is to identify, characterize and documentthe therapeutic potential of the TREM-1 derived peptide hP5, in anexperimental model of colitis in mice.

On day 0 of the study, the water supply was removed and replaced with 3%Dextran Sulfate Sodium (DSS) From day 0 to day 6, this solution was theonly source of fluids. Water was administered for the rest of theexperiment (day 7-11). Healthy controls received water only.

From day 3 to day 10 mice were treated with either hP5, or asequence-scrambled control peptide. Peptides were dissolved in DMSO at10 mg/ml and stored at +4° C. Before administration, the stock solutionwas diluted 1:10 in water 10% Solutol® HS 15 (BASF). Final vehicleconcentrations: 1% DMSO, 10% solutol in water. 200 μl of these freshlyprepared solutions of hP5, its scrambled control or vehicle wereinjected intraperitoneally with a 25 G needle.

Animals were weighted daily and monitored on days 3, 4, 5, 6, 7 and 10for rectal bleeding and stool consistency. For each group, the diseaseactivity index (DAI) was determined by evaluating changes in weight,stool consistency and presence of gross blood during the study, asdescribed below:

Weight loss Blood in Score (%) Stool consistency stool 0  <1 NormalNegative 1 1-4, 9 Soft +/− 2 5-9, 9 Mixed (soft and liquid) + 3 10-15Liquid ++ 4 >15 Diarrhea (liquid stools that adhere to Gross the anus)bleeding

Scoring System for the Disease Activity Index (DAI).

Individual scores for each parameter are added and then divided by threeto give a DAI score for each mouse.

To determine the presence of occult blood in stool, a pea-sized stoolsample was placed on a slide. Then two drops of reagent (Hemocult Sensa,Beckman Coulter) were placed onto the stool sample on the slide and achange of colour was observed. The presence of occult blood was gradedusing a score of 0 for no colour; 1 for a very light blue (+/−) colourtaking over 30 seconds to appear, 2 for a blue colour developing in 30seconds or more (+); 3 for an immediate change in colour (++) and 4 forgross blood observable on the slide.

On day 11 mice were euthanized by cervical dislocation to allow colonlength evaluation. An incision was done in the abdomen to expose thecolon. The stool in the colon was removed flushing with saline. Theentire colon from cecum to anus was removed and the length was measuredand reported as colon length (cm).

Data were analyzed using Graph Pad Prism. Results are given asmeans±standard error of the mean. The BW score, stool score and bloodscore were analyzed using two-way ANOVA test, Bonferroni post test.Colon length was analysed using one-way ANOVA, Dunn's post test.*P≦0.05; **P≦0.01; ***P≦0.001

mP5 is a mTREM-1 derived peptide whose efficacy has been proven inseveral models of sepsis. In this study, the investigators have testedthe efficacy of the human orthologue of mP5, compared to its scrambledpeptide control, in the above described DSS-induced colitis model. Allpeptides were administered in a therapeutic fashion, 3 days afterinitiation of DSS treatment.

In all animals, body weight, haemoccult or presence of gross blood andstool consistency were monitored. Bloody stools were observed from day 3onwards, loose stools and weight loss appeared beginning from day 4-5.These comprehensive functional measures, that were somewhat analogous toclinical symptoms observed in human IBD, are summarized by the DiseaseActivity Index, as shown in FIG. 20. This scoring method, validated byrepeated studies, showed minimum variations and correlates well withmore specific measures of inflammation.

None of the control animals showed disease activity. DAI peaked at day5-6 and regressed upon DSS removal. As soon as colitis was established(day 5), hP5 administration significantly ameliorated stool consistency(FIG. 23) and colon bleeding (FIG. 22), as shown by approximately a 50%DAI inhibition, when compared to mice treated with DSS+vehicle orDSS+scrambled peptide (1.95±0.170 vs. 2.92±0.054, p≦0.001 or 3.00±0.088,p≦0.001, respectively).

Protection lasted during the whole treatment. Notably at day 10, in thehP5-treated group both stool score and hemoccult were normal (FIGS. 23and 22), while the groups treated with DSS+vehicle and DSS+scrambledpeptide still showed clinical signs of inflammation (blood score:DSS+hP5=0.43±0.202 vs DSS+vehicle=1.25±0.164; p≦0.01; DSS+hP5=0.43±0.202vs DSS+scrambled peptide=1.75±0.250; p≦0.001. Stool score:DSS+hP5=0.71±0.474 vs DSS+vehicle=1.75±0.250 p≦0.05; DSS+hP5=0.71±0.474vs DSS+scrambled peptide=2.38±0.324; p≦0.001).

We also monitored weight loss associated with the development ofcolitis. FIG. 21 represents the percentage of weight loss, expressed asa score. Mice started loosing weight at day 4 and the maximal weightloss was reached at day 8. hP5 treatment significantly reduced weightloss in comparison with DSS+scrambled, from day 8 on (day 10:hP5=2.00±0.436 vs DSS+scrambled pep. 3.50±0.189; p≦0.001). At day 11mice were euthanized and the colon length of each mouse measured fromthe anus to the end of the cecum (FIG. 24). DSS-induced coloninflammation caused a 30% colon shortening when compared to naïveanimals. In the hP5-treated group, colon shortening was significantlyameliorated when compared to control group (hP5=7.41±0.237 vsDSS+vehicle=5.96±0.230; p≦0.05). In conclusion, blocking TREM-1 with ahuman TREM-1 derived peptide attenuates intestinal inflammation evenwhen the peptide is administered after the appearance of the clinicalsigns of colitis. This finding indicates that the human TREM-1-derivedpeptide efficiently blocks interaction of the mouse TREM-1 receptor withits endogenous ligand.

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The invention claimed is:
 1. An isolated peptide or derivative thereof,which is capable of acting as an antagonist of the TREM-1 protein asdefined by SEQ ID NO. 1, comprising the amino acid sequence of SEQ IDNO. 20 or at least 3 amino acids of SEQ ID NO. 21, wherein: (a) thepeptide consists of: (i) a contiguous sequence of 5 to 29 amino acidsfrom SEQ ID NO: 1; or (ii) a contiguous sequence of 5 to 29 amino acidsfrom SEQ ID NO: 1 in which one amino acid is substituted conservativelywith another amino acid; or (b) the peptide consists of an amino acidsequence having at least 80% sequence identity to SEQ ID NOs: 16, 17, 18or 19; and wherein the derivatives of the peptides of (a) or (b) aremodified by glycosylation, acetylation, pegylation, phosphorylation,amidation, or derivatization by protecting or blocking groups.
 2. Theisolated peptide or derivative thereof according to claim 1, whichconsists of: (a) a contiguous sequence of 5 to 29 amino acids from SEQID NO: 1; or (b) a contiguous sequence of 5 to 29 amino acids from SEQID NO: 1 in which one amino acid is substituted conservatively withanother amino acid; and wherein the derivatives of the peptides of (a)or (b) are modified by glycosylation, acetylation, pegylation,phosphorylation, amidation, or derivatization by protecting or blockinggroups.
 3. The isolated peptide or derivative thereof according to claim1, wherein the peptide consists of an amino acid sequence having atleast 80% sequence identity to SEQ ID NOs: 16, 17, 18 or 19; and whereinthe derivatives of the peptide are modified by glycosylation,acetylation, pegylation, phosphorylation, amidation, or derivatizationby protecting or blocking groups.
 4. The isolated peptide or aderivative thereof according to claim 3, wherein the peptide consists ofan amino acid sequence having at least 80% sequence identity to SEQ IDNO:
 19. 5. The isolated peptide or derivative thereof according to claim3, wherein the peptide consists of an amino acid sequence having thesequence of SEQ ID NOs: 16, 17, 18 or 19, or which differs from saidsequence by one or more conservative amino acid modifications.
 6. Theisolated peptide according to claim 4, wherein the peptide consists ofan amino acid having the sequence of SEQ ID NO: 19, or which differsfrom said sequence by one or more conservative modifications.
 7. Theisolated peptide or derivative thereof according to claim 1, whereinsaid peptide consists of a contiguous sequence of 15 to 29 amino acidsfrom SEQ ID NO.
 1. 8. The isolated peptide or derivative thereofaccording to claim 1, which comprises at least 3 amino acids from SEQ IDNO. 21, wherein the at least 3 amino acids from SEQ ID NO. 21 areselected from the group consisting of QPP, QPPK (SEQ ID NO: 24), andQPPKE (SEQ ID NO. 21).
 9. The isolated peptide or derivative thereofaccording to claim 3, wherein the peptide consists of an amino acidsequence having the sequence of SEQ ID NOs: 16, 17, 18 or
 19. 10. Acomposition comprising the isolated peptide or derivative thereofaccording to claim 1, and a pharmaceutically acceptable carrier.